Role of myocardial 5-HT4 and 5-HT2A receptors in heart failure



Effects of serotonin in failing cardiac ventricle – signalling mechanisms and potential therapeutic implicationspossible therapeutic opportunities

Running title: 5-HT receptors in failing cardiac ventricle

Finn Olav Levy*, Eirik Qvigstad, Kurt A. Krobert, Tor Skomedal, Jan-Bjørn Osnes

Department of Pharmacology and Centre for Heart Failure Research, Faculty of Medicine, University of Oslo, Oslo, Norway

*Correspondence: Finn Olav Levy, Department of Pharmacology, University of Oslo, P.O. Box 1057 Blindern, N-0316 Oslo, Norway.

Phone: +47-22840237/01, Fax: +47-22840202, E-mail: f.o.levy@medisin.uio.no

Keywords

5-HT2A receptor; 5-HT4 receptor; Heart failure; Inotropic effect; Ventricle; Heart

SummaryAbstract

Previously, cardioexcitation by serotonin (5-hydroxytryptamine, 5-HT) was believed to be confined to atria in mammals including man, and mediated through 5-HT4 receptors in pig and man, but 5-HT2A receptors in rat. Recent studies, reviewed here, demonstrate that functional 5-HT4 receptors can be revealed in porcine and human ventricular myocardium during phosphodiesterase inhibition, and that 5-HT4 receptor mRNA is increased in human heart failure. In rats, functional cardiac ventricular 5-HT4 and 5-HT2A receptors appear in the cardiac ventricle during heart failure and mediate inotropic responses through different mechanisms. 5-HT2A receptor signalling resembles that from (1-adrenoceptors and causes inotropic effects through increased myosin light chain phosphorylation, resulting in Ca2+ sensitisation. 5-HT4 receptor signalling resembles that from (-adrenoceptors and causes inotropic effects through a pathway involving cAMP and PKA-mediated phosphorylation of proteins involved in Ca2+ handling, resulting in enhanced contractility through increased Ca2+ availability. Cyclic AMP generated through 5-HT4 receptor stimulation seems more efficiently coupled to increased contractility than cAMP generated through (-adrenoceptor stimulation. Increasing contractility through cAMP is considered less energy efficient than Ca2+ sensitisation and this may be one reason why (-adrenoceptor antagonism is beneficial in heart failure patients. Treatment of heart failure rats with the 5-HT4 antagonist SB207266 (piboserod) resulted in potentially beneficial effects, although small. Further studies are needed to clarify if such treatment will be useful for patients with heart failure.

Introduction

Serotonin is a cardioexcitatory neurotransmitter in the ventricle of some primitive organisms, e.g. aplysia (Sawada et al., 1984). However, in mammals, cardioexcitatory effects of serotonin have been considered to be restricted to the atria (Kaumann and Sanders, 1998). Possibly reflecting the ancient evolutionary origin of cardiac serotonin receptors and serotonin responses, the excitatory effects of serotonin are mediated by different receptors in different species, e.g. 5-HT2A in rat, 5-HT3 (through noradrenaline release) in rabbit, 5-HT3 and 5-HT4 in guinea pig and 5-HT7 in cat (see Kaumann and Sanders (1998) for references). In pig and man, 5-HT4 receptors are present in atria, and a role for these receptors in atrial fibrillation has been suggested (Kaumann, 1994) and supported by experimental models (Rahme et al., 1999). The idea that porcine and human 5-HT4 receptors were restricted to atria, gained support from several studies concluding that 5-HT4 receptor stimulation did not have effects on porcine (Lorrain et al., 1992; Saxena et al., 1992; Schoemaker et al., 1992) or human (Jahnel et al., 1992; Schoemaker et al., 1993) ventricle. However, several recent reports, reviewed below, have challenged the view that cardioexcitatory effects of serotonin are restricted to atria, particularly in heart failure, where ventricular inotropic responsiveness to serotonin becomes apparent, either through 5-HT4 receptors and or through 5-HT2A receptors or through both, depending on species and stage of heart failure.

Heart failure can be considered a final common pathwayend-point of cardiac disease, where the diseased heart is no longer able to meet the circulatory requirements of the body, due to myocardial infarction or other causes. This leads to several compensatory mechanisms, which are now generally considered to be of potential short term benefit, but long term disadvantage. Thus, modern pharmacological treatment of chronic heart failure is generally aimed at opposing these compensatory mechanisms. Examples of such treatment which has proven to reduce mortality in heart failure is (-adrenoceptor antagonism (Tendera and Ochala, 2001; Cleland, 2003), angiotensin converting enzyme (ACE) inhibition/angiotensin receptor antagonism (Poole-Wilson, 2003) and aldosterone receptor antagonism (Rajagopalan and Pitt, 2003). Whether serotonin receptor antagonism can be added to this list remains an open question and will be discussed below.

Of note, it is believed that some compensatory mechanisms are more deleterious than others. This is best illustrated by increased neurohormonal activation of the adrenergic system during heart failure, where (-adrenoceptors and (1-adrenoceptors utilise completely different signalling mechanisms to achieve increased contractility. Whereas (-adrenoceptors through increased cAMP levels cause increased contractility mainly through increased Ca2+ handling, (1-adrenoceptor stimulation has recently been shown to increase phosphorylation of regulatory myosin light chain (MLC-2), thought to increase contractility through Ca2+ sensitisation (Andersen et al., 2002). Whereas increasing contractility through (-adrenoceptor stimulation and thus increased Ca2+ handling is quite energy-consuming, it appears that increasing contractility through (1-adrenoceptor stimulation and thus increased Ca2+ sensitivity is more energy efficient (Hasenfuss et al., 1989). This may contribute to explainbe a reason why (-adrenoceptor antagonism in heart failure is beneficial (Lohse et al., 2003), whereas (1-adrenoceptor antagonism is not (Osnes et al., 2000). Relating this to serotonin receptors, it is worth noting that 5-HT4 receptors and (-adrenoceptors both signal through Gs and increased cAMP formation, whereas 5-HT2A and (1-adrenoceptors are both classically considered as receptors which activate similar signalling pathways, by coupling to the G proteins Gq/11 and probably G12/13 (Fig. 1).

5-HT4-mediated effects in porcine and failing human ventricle

The first demonstration of 5-HT4 mRNA expression in mammalian ventricle was published by Bach et al. (2001), who reported expression of 5-HT4(a) and 5-HT4(b) receptor mRNA in human left and right cardiac ventricle in post-mortem material from two persons with no reported history of heart failure. Previous reports had failed to detect 5-HT4(a) or 5-HT4(b) receptor mRNA in human ventricle (Blondel et al., 1997; Blondel et al., 1998) and had failed to detect cardiostimulant effects of 5-HT in isolated human ventricular muscle (Jahnel et al., 1992; Schoemaker et al., 1993). Brattelid et al. (2004b) later reported functional 5-HT4 receptors in porcine as well as human ventricular myocardium with increased 5-HT4 mRNA levels in human failing left and right ventricle. In the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), serotonin increased contractile force and PKA activity in ventricular trabeculae from both newborn piglets and adult pigs. The potency and efficacy of serotonin to increase contractile force as well as its efficacy to increase PKA activity was higher in adult pigs than piglets. In failing human hearts, serotonin effects were generally increased by PDE inhibition with IBMX. In the absence of PDE inhibition, serotonin increased force in some, but not all trabeculae tested. In the presence of IBMX, serotonin consistently increased contractile force and hastened relaxation, as well as caused arrhythmias in some trabeculae. All serotonin effects were blocked by 5-HT4 receptor antagonists. Thus, PDE inhibition uncovers functional 5-HT4 receptors, coupled to a PKA pathway, through which serotonin enhances contractility, hastens relaxation and can potentially cause arrhythmias. 5-HT4 receptor mRNA was expressed in both porcine and human ventricles, and increased four-fold in 20 failing human hearts compared to five donor hearts.

5-HT4-mediated effects in chronic failing rat ventricle

In rats, serotonin caused positive inotropic and lusitropic effects in papillary muscles from rats with chronic congestive heart failure (CHF) 6 weeks after a large myocardial infarction induced by coronary artery ligation, but not from sham-operated controls (Qvigstad et al., 2005a). The finding was surprising, since functional 5-HT4 receptors had previously not been detectable in rat heart, even in the atria, despite the presence of 5-HT4 receptor mRNA (Läer et al., 1998). The inotropic effect of 10 (M serotonin in CHF was of comparable size as that of (-adrenoceptor stimulation with 10 (M isoproterenol (31±2% vs. 34±2% measured as increase in Fmax, and 45±3% vs. 59±7% measured as increase in (dF/dt)max). The serotonin effects were antagonised by GR113808 with high affinity (-logK=9.5), consistent with mediation through 5-HT4 receptors. Minor positive inotropic effects as well as partial blockade of 5-HT-mediated effects were also observed by the 5-HT4-selective partial agonist RS67506, as expected for a 5-HT4-mediated response. The serotonin effects were blunted by carbachol, and serotonin increased cardiomyocyte and whole ventricular cAMP levels, consistent with coupling to Gs and adenylyl cyclase. 5-HT4(b) receptor mRNA expression increased four-fold in left ventricle and 18-fold in papillary muscle of CHF vs. Ssham-operated rats, as measured by real-time quantitative RT-PCR. The 5-HT4-mediated inotropic response and 5-HT4 mRNA levels increased with increasing myocardial infarction, also in rats not developing CHF. Thus, myocardial infarction and CHF cause the appearance of functional 5-HT4 receptors in the rat ventricle. This represents a novel model for ventricular 5-HT4 receptors of human failing heart, which will enable further studies of the role of 5-HT4 serotonin receptors in heart failure.

5-HT2A-mediated effects in rat and human ventricle

It turned out that responsiveness to serotonin appeared early after myocardial infarction in the rat ventricle and was apparent already one day after infarction in rats with severe acute heart failure (Qvigstad et al., 2005c). It was observed that the potency of serotonin to stimulate an inotropic response was lower in acute (up to about 7 days) than chronic (6 weeks) heart failure. Since it is known that the affinity of serotonin for 5-HT2A receptors is rather low, this led Qvigstad et al. (2005c) to explore whether 5-HT2A receptors could also be involved, possibly in addition to 5-HT4 receptors, in mediating an inotropic response in acute heart failure. By the use of the 5-HT4-selective antagonist GR113808 and the relatively selective 5-HT2A antagonist ketanserin, it was clearly demonstrated that the inotropic response to serotonin in acute heart failure (3 days after coronary artery ligation and myocardial infarction) was a mixed response, mediated by both 5-HT4 and 5-HT2A receptors. Consistent with the 5-HT2A-mediated inotropic response, increased expression of 5-HT2A receptor mRNA was demonstrated in left ventricle of rats with acute CHF heart failure by real-time quantitative RT-PCR. Although it is not yet known if 5-HT2A receptors mediate inotropic responses in human failing ventricle, increased 5-HT2A receptor mRNA expression was recently demonstrated in human failing ventricle (Brattelid et al., 2007).

How does 5-HT2A receptor stimulation increase cardiac contractility?

Whereas the 5-HT4-mediated inotropic response (studied in the presence of ketanserin) developed rapidly and was fully developed in about 1 min after serotonin addition, the 5-HT2A-mediated inotropic response (studied in the presence of GR113808) developed more slowly and was not fully developed until more than 5 min after addition of serotonin (Qvigstad et al., 2005c). Further studies revealed that the 5-HT2A-mediated inotropic response was triphasic, consisting of an initial positive component, a transient negative component and a slower, larger and sustained positive component. Detailed studies of this triphasic response, utilising inhibitors of different intracellular signalling pathways, revealed that the triphasic pattern resulted from a positive inotropic effect and a transient negative inotropic effect with different kinetics.

As 5-HT2A receptors activate similar G proteins (Gq/11 and probably G12/13) as (1-adrenoceptors in other tissues, Qvigstad et al. (2005c) examined whether 5-HT2A receptor stimulation could mediate increased contractility through increasing the phosphorylation of regulatory myosin light chain (MLC-2), as was recently shown in our lab for (1-adrenoceptor-mediated inotropic response (Andersen et al., 2002). The positive inotropic 5-HT2A response was accompanied by increased MLC-2 phosphorylation and was sensitive to the MLC kinase inhibitor ML-9, the calmodulin inhibitor W-7 and the Rho kinase (ROCK) inhibitor Y-27632, all consistent with a central role of myosin light chain phosphorylation in the inotropic response. 5-HT2A receptor stimulation also induced a positive inotropic response (of 18%) in isolated cardiomyocytes from chronic failing ventricle, 6 weeks after myocardial infarction (Birkeland et al., 2007b). Although a minor increase in Ca2+ transient was observed (6%), 5-HT2A receptor stimulation did not increase ICa,L, SR Ca2+ content, phospholamban serine16 (PLB-Ser16) or troponin I phosphorylation, but increased myosin light chain (MLC-2) phosphorylation. However, the exact signalling mechanisms from 5-HT2A receptor stimulation to MLC phosphoryation remain to be elucidated. From our findings, the increased MLC phosphorylation probably results from a combination of increased MLC kinase activity and reduced MLC phosphatase activity (Fig. 1). Whether the changes of these enzyme activities occur simultaneously or sequentially, is an open question.

The transient negative effect was eliminated by the protein kinase C (PKC) inhibitor bis-indolyl-maleimide (BIM), which inhibits all PKC isoforms. Further studies using the relatively isoform-selective PKC inhibitors Gö6976 and Rottlerin indicated that the most likely PKC isoform to mediate the transient negative effect is PKC( (Qvigstad et al., 2005c).

Combined 5-HT2A and 5-HT4-mediated inotropic responses in afterload-induced cardiac hypertrophy and failure

From the studies of rats with myocardial infarction and post-infarction heart failure outlined above, it could not be inferred that serotonin responsiveness would also become apparent in the ventricles of rats with heart failure from other causes. It was also unknown whether cardiac hypertrophy would be associated with ventricular serotonin responsiveness. This was addressed in a recent study (Brattelid et al., 2007), where rats were subjected to banding of the ascending aorta to increase cardiac afterload, thereby inducing left ventricular hypertrophy and eventually heart failure in the most severely affected animals. The rats were studied six weeks after aortic banding, by measuring inotropic responses in left ventricular papillary muscles as well as serotonin receptor mRNA expression by real-time RT-PCR. The study revealed robust inotropic responses to serotonin through both 5-HT4 and 5-HT2A receptors in both rats with ventricular hypertrophy without heart failure and in rats with heart failure. Whereas no 5-HT4-mediated inotropic response was detected in the sham-operated animals, these animals did show a measurable inotropic response to 5-HT2A stimulation. The study demonstrated a differential regulation of 5-HT2A- and 5-HT4-mediated inotropic responsiveness. The 5-HT4-mediated inotropic response was induced in hypertrophy and was further increased in the rats with left ventricular dilation and heart failure. The 5-HT4-mediated inotropic response correlated well with 5-HT4 mRNA expression. On the other hand, the 5-HT2A-mediated inotropic response increased with increasing degree of hypertrophy, but did not increase further in the heart failure group. Furthermore, the 5-HT2A-mediated inotropic response did not correlate well with 5-HT2A mRNA expression. The study also demonstrated increased 5-HT2B receptor mRNA expression in failing ventricle, but this increase appeared confined to non-cardiomyocytes, and the 5-HT2B receptor did not mediate any inotropic response.

Functional 5-HT2A and 5-HT4 receptors are expressed in cardiomyocytes

Since stimulation of 5-HT2A and 5-HT4 receptors led to positive inotropic effects in papillary muscles and ventricular muscle strips, it could be inferred that these receptors must be localised on cardiomyoctes, as opposed to other cell types present in these preparations. Additional functional evidence that both these receptors are actually localised on the cardiomyocytes was obtained by Birkeland et al. (2007b), who documented electrophysiological and contractile responses to stimulating these receptors in isolated, single cardiomyocytes from rats with heart failure 6 weeks after myocardial infarction. On the other hand, 5-HT2B receptors, which have been implicated in cardiac development (Nebigil et al., 2000) and have been artificially expressed in cardiomyocytes (Nebigil et al., 2003) seem to play no role in the inotropic response to serotonin and to occur naturally mainly on non-cardiomyocytes, based on the results of Brattelid et al. (2007), who demonstrated that increased 5-HT2B receptor mRNA expression in failing ventricle appeared confined to non-cardiomyocytes and that the 5-HT2B receptor did not mediate any inotropic response.

Does 5-HT4 and 5-HT2A expression in heart failure reflect reactivation of a foetal gene program?

One characteristic of the failing ventricle is a transition towards a foetal gene expression pattern (Chien et al., 1991). To explore the role of 5-HT4, 5-HT2A and 5-HT2B receptors in the foetal heart, Brattelid et al. (2006) collected ventricle from Wistar rats 3 days and 1 day before expected birth (days -3 and -1), as well as 1, 3, 5 and 113 days after birth, and measured expression of 5-HT4, 5-HT2A and 5-HT2B receptor mRNA by real-time quantitative RT-PCR as well as contractile function mediated by these receptors ex vivo. Both 5-HT4 mRNA expression and function was found to be highest at day -3 and decreased gradually from day -3 to day 5, with a further decrease to adult levels (day 113). The levels increased in CHF, but the levels seen in the foetal ventricle were higher than the levels in CHF. The 5-HT2A and 5-HT2B receptor mRNA levels increased to a maximum immediately after birth, a situation associated with an acute increase in wall stress, but only the 5-HT2A receptor mediated a ventricular inotropic response. Based on these results, the 5-HT4 receptor may be a representative of the foetal cardiac gene program and play a significant role in early cardiac development as well as CHF. Similarly, the 5-HT2A and 5-HT2B receptors might play important roles in postnatal heart development associated with an acute increase in wall stress and subsequent hypertrophy. In the mouse, there is also evidence of foetal cardiac 5-HT4 receptor expression, which declines towards birth (Kamel et al., 2007).

How similar is 5-HT4 receptor stimulation and (-adrenoceptor stimulation in the failing ventricle?

Both 5-HT4 receptors and (-adrenoceptors are coupled to the G protein Gs. Thus, it is to be expected that stimulation of these receptors will have similar effects in the failing ventricle. Although more than one mechanism of action may be involved in the 5-HT4-mediated inotropic response, several lines of evidence indicate that the ventricular inotropic effect of serotonin is at least partly mediated through cAMP (Qvigstad et al., 2005a; Brattelid et al., 2004b; Qvigstad et al., 2005b): (1) Serotonin increased cAMP levels, although to a small extent, in perfused hearts and isolated cardiomyocytes. (2) Serotonin elicited fast-developing inotropic and lusitropic effects – characteristics similar to responses to (-adrenoceptor stimulation by isoproterenol. (3) The inotropic effect of serotonin was attenuated by muscarinic receptor activation. (4) In isoproterenol-stimulated muscle, serotonin did not cause additional response, as would be expected if different signalling pathways were used. (5) In failing human ventricle, the maximal inotropic and lusitropic responses to serotonin were enhanced by phosphodiesterase inhibition. (6) In porcine ventricle, 5-HT4 receptor stimulation resulted in PKA activation. Thus, all the functional and pharmacological characteristics observed in relation to the serotonin response are as expected for cAMP-mediated mechanical responses in mammalian heart muscle (Skomedal et al., 1997). This was further addressed in a recent study of cardiomyocytes isolated from failing rat ventricle, 6 weeks after myocardial infarction (Birkeland et al., 2007b), where contractile function and Ca2+ transients were measured in field stimulated cardiomyocytes, and L-type Ca2+-current (ICa,L) and sarcoplasmic reticulum (SR) Ca2+ content were measured in voltage-clamped cells. 5-HT4 receptor stimulation induced a positive inotropic response of 33%, and also increased the Ca2+ transient by 44%. ICa,L and SR Ca2+ content were increased by 57% and 65%, and phospholamban serine16 (PLB-Ser16) and troponin I phosphorylation increased by 26% and 13% after 5-HT4 receptor stimulation, as measured by Western blotting. Thus, all the data so far indicate that 5-HT4 receptors in the rat (as well as human and porcine) ventricles utilise similar signalling mechanisms as 5-HT4 receptors in human atria, which increase cAMP levels, accompanied by increased PKA activity and phosphorylation of L-type calcium channels, phospholamban and troponin-I, similar to (-adrenoceptor activation (Kaumann and Sanders, 1998; Kaumann and Levy, 2006; Kaumann et al., 1990).

However, some data may indicate that cAMP resulting from stimulation of 5-HT4 receptors is more efficiently coupled to functional responses than cAMP resulting from stimulation of (-adrenoceptors. For example, Qvigstad et al. (2005a) found that even though stimulation of 5-HT4 receptors and (-adrenoceptors (by isoproterenol) in CHF papillary muscle resulted in essentially similar inotropic responses, the increase in cAMP levels measured in perfused CHF hearts following stimulation with serotonin was considerably lower than following isoproterenol stimulation. This may reflect a differential compartmentation of cAMP-mediated signalling following stimulation of 5-HT4 and (-adrenoceptors. Phosphodiesterases (PDEs) play important roles in compartmentalised cAMP/PKA signalling (Beavo and Brunton, 2002). Afzal et al. (2007) recently addressed which PDEs contribute to limit the inotropic response to 5-HT4 receptor stimulation in post-infarction rat and failing human hearts and found that PDE3 is the main PDE regulating this response and that an involvement of PDE4 is demasked by concomitant inhibition of PDE3. A direct comparison with the different (-adrenoceptors is not yet available, as most studies on PDE involvement in compartmentation of (-adrenoceptor signalling have either involved measurements of other parameters than inotropic response, e.g. measurement of localised intracellular cAMP pools (Mongillo et al., 2004; Rochais et al., 2006) or have used different model systems, e.g. neonatal cardiomyocytes (Xiang et al., 2005). When comparing 5-HT4 signalling to (-adrenoceptor signalling, it will also be necessary to differentiate between signalling via (1-adrenoceptors and (2-adrenoceptors, as illustrated by two recent studies, which indicate differential effects of PDE3 and PDE4 on β1- and β2-adrenoceptor-mediated effects in human atrium (Christ et al., 2006) as well as mouse heart (Galindo-Tovar and Kaumann, 2008), and even between different functional states of the (1-adrenoceptor (Vargas et al., 2006).

Is 5-HT4 receptor stimulation deleterious in CHF, and could blockade of these receptors represent a new treatment modality?

Since (-adrenoceptor-mediated signalling is deleterious in heart failure, and given that the mechanism of stimulating ventricular inotropic response is similar between 5-HT4 receptors and (-adrenoceptors, a natural question to ask is whether 5-HT4 receptor stimulation is also deleterious in CHF. So far, all clinical trials with cAMP-enhancing agents in chronic heart failure, including phosphodiesterase inhibitors, have adversely affected survival (Amsallem et al., 2005). Even apparently weak stimulation of cardiac cAMP levels may be detrimental in heart failure, as demonstrated by the detrimental effect of the partial beta-adrenoceptor agonist xamoterol in human heart failure clinical trials (The Xamoterol in Severe Heart Failure Study Group, 1990). An inotropic activity of xamoterol comparable to the inotropic effect we observed with serotonin in failing human ventricle (Brattelid et al., 2004b) was demonstrated on isolated human myocardial preparations only in the presence of the phosphodiesterase inhibitor milrinone or the adenylyl cyclase stimulator forskolin (Böhm et al., 1990).

One way to address the question if 5-HT4 receptor stimulation is deleterious in heart failure would be to test whether treatment with a 5-HT4 receptor antagonist is beneficial in heart failure. Such an experiment was performed by Birkeland et al. (2007a) by treatment of rats with post-infarction heart failure with the 5-HT4 antagonist piboserod (SB207266). Rats were subjected to ligation of the left coronary artery, and rats with large infarction as assessed by echocardiography three days after surgery were randomised to six-week treatment with piboserod, delivered via subcutaneous mini-osmotic pumps at a rate of 0.5 mg/kg/24h or corresponding placebo. After six weeks the rats in the piboserod group, compared to the placebo group, had improved diastolic function, with 4.6% lower left ventricular diastolic diameter and 24% lower mitral flow deceleration (reflecting better compliance), consistent with reduced remodelling as seen after treatment with (-adrenoceptor-antagonists (“beta-blockers”) (Omerovic et al., 2003; Doughty et al., 2004). The rats in the piboserod group also had reduced left ventricular systolic diameter (by 6.1%), reduced heart weight (by 10.2%) and reduced lung weight (by 13.1%). The changes in posterior wall thickening and shortening velocity, cardiac output, left ventricular systolic pressure and (dP/dt)max, parameters of left ventricular systolic function, did not reach statistical significance. Two parameters possibly reflecting neurohumoral activation in heart failure were also measured. The positive inotropic response to isoproterenol, which decreases in heart failure, was partially restored towards normal by treatment with piboserod (36% increase compared to placebo). Furthermore, the 5-HT4-mediated positive inotropic response to serotonin, which appears in this model of heart failure, was decreased by 57% by treatment with piboserod compared to placebo. Both these changes could reflect a partial normalisation, reflecting improvement of the heart failure. Since it could be argued that the decrease in 5-HT4-mediated inotropic response in the piboserod group could reflect residual piboserod blocking the 5-HT4 receptors, measures were taken to avoid this, such as an extensive wash-out period of the papillary muscles before the measurements were started and use of suffiently high concentration of serotonin to reach maximal response (Birkeland et al., 2007a).

The finding that chronic treatment with a 5-HT4 antagonist had effects on ventricular function in heart failure, although modest, would seem to indicate that cardiac ventricular 5-HT4 receptors are indeed stimulated by endogenous serotonin. Possible sources of serotonin include the heart tissue itself, nerve endings and platelets. The human heart (left ventricular papillary muscle) contains about 400 ng serotonin/g tissue, but its histological localisation, synthesis, metabolism and release mechanisms are unknown (Sole et al., 1979). A possible source of serotonin within the heart may be mast cells, which are found in the heart (Dvorak, 1986) and which have the capacity to store and release serotonin (Fuder et al., 1994). A role for mast cells in heart failure has been proposed previously (Hara et al., 2002), although it is as yet unknown whether this would also involve serotonin. Capture of serotonin and release by sympathetic nerve endings of human atrium have been shown experimentally to enhance contractile force through 5-HT4 receptors (Kaumann, 2000). Plasma serotonin, in part derived from platelets, was found to be increased in CHF patients (Chandra et al., 1994; Vizir and Berezin, 2001). Serotonin released from aggregating platelets could also conceivably reach the myocardium under conditions of endocardial damage (Shah et al., 1989). Alternatively, since it has been reported that 5-HT4 receptors display constitutive activity and piboserod (SB207266) in vitro is an inverse agonist (Claeysen et al., 1999; Brattelid et al., 2004a), the effects of treatment with piboserod could be explained by antagonising the constitutive activity of cardiac 5-HT4 receptors. However, such an explanation would require rigorous proof of the lack of available endogenous agonist. An alternative interpretation of the apparent effectiveness of 5-HT4 receptor antagonism in heart failure could come from the thorough studies by the group of Lefebvre et al., demonstrating that serotonin through 5-HT4 receptors contributes to increase aldosterone secretion by the adrenal glands (Lefebvre et al., 1998). In this context, it could be interesting to test if the effects of 5-HT4 receptor antagonism in heart failure are also present on top of treatment with aldosterone antagonist, e.g. spironolactone or eplerenone.

It is possible, as suggested by Birkeland et al. (2007a), that 6-week treatment with piboserod was not sufficiently long to reveal the full potential of 5-HT4 antagonism in rats with heart failure. A study of longer duration and a study comparing the effects of treatment with 5-HT4 antagonist and (-adrenoceptor antagonist separately and in combination would be of interest to further clarify the usefulness of 5-HT4 receptor antagonism in heart failure. A clinical study assessing the potential of piboserod for the treatment of heart failure patients has also been performed (), although the results are not yet published. The results of this study will hopefully indicate whether 5-HT4 antagonism can become a useful addition to the armamentarium of drugs used for treatment of heart failure.

Acknowledgements

Work in the authors’ laboratories are funded by grants from The Research Council of Norway, The Norwegian Council on Cardiovascular Diseases, Anders Jahre’s Foundation for the Promotion of Science, The Novo Nordisk Foundation, The Family Blix Foundation and the University of Oslo.

References

Afzal, F., Andressen, K. W., Mørk, H. K., Aronsen, M., Moltzau, L. R., Sjaastad, I., Skomedal, T., Levy, F. O., Osnes, J. B., Qvigstad, E., 2007. 5-HT4-mediated positive inotropic effect is regulated by phosphodiesterase 3 in failing rat heart and human ventricle. J. Mol. Cell. Cardiol. 42/S1, S152.

Amsallem, E., Kasparian, C., Haddour, G., Boissel, J. P., Nony, P., 2005. Phosphodiesterase III inhibitors for heart failure. Cochrane. Database. Syst. Rev., CD002230.

Andersen, G. Ø., Qvigstad, E., Schiander, I., Aass, H., Osnes, J. B., Skomedal, T., 2002. a1-AR-induced positive inotropic response in heart is dependent on myosin light chain phosphorylation. Am. J. Physiol Heart Circ. Physiol 283, H1471-H1480.

Bach, T., Syversveen, T., Kvingedal, A. M., Krobert, K. A., Brattelid, T., Kaumann, A. J., Levy, F. O., 2001. 5-HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle. Naunyn-Schmiedeberg's Arch. Pharmacol. 363, 146-160.

Beavo, J. A., Brunton, L. L., 2002. Cyclic nucleotide research - still expanding after half a century. Nat. Rev. Mol. Cell Biol. 3, 710-718.

Birkeland, J. A., Sjaastad, I., Brattelid, T., Qvigstad, E., Moberg, E. R., Krobert, K. A., Bjørnerheim, R., Skomedal, T., Sejersted, O. M., Osnes, J. B., Levy, F. O., 2007a. Effects of treatment with a 5-HT4 receptor antagonist in heart failure. Br. J. Pharmacol. 150, 143-152.

Birkeland, J. A., Swift, F., Tovsrud, N., Enger, U., Lunde, P. K., Qvigstad, E., Levy, F. O., Sejersted, O. M., Sjaastad, I., 2007b. Serotonin increases L-type Ca2+ current and SR Ca2+ content through 5-HT4 receptors in failing rat ventricular cardiomyocytes. Am. J. Physiol Heart Circ. Physiol 293, H2367-H2376.

Blondel, O., Gastineau, M., Dahmoune, Y., Langlois, M., Fischmeister, R., 1998. Cloning, expression, and pharmacology of four human 5-hydroxytryptamine4 receptor isoforms produced by alternative splicing in the carboxyl terminus. J. Neurochem. 70, 2252-2261.

Blondel, O., Vandecasteele, G., Gastineau, M., Leclerc, S., Dahmoune, Y., Langlois, M., Fischmeister, R., 1997. Molecular and functional characterization of a 5-HT4 receptor cloned from human atrium. FEBS Lett. 412, 465-474.

Böhm, M., Mittmann, C., Schwinger, R. H., Erdmann, E., 1990. Effects of xamoterol on inotropic and lusitropic properties of the human myocardium and on adenylate cyclase activity. Am. Heart J. 120, 1381-1392.

Brattelid, T., Bekkevold, S. V. S., Qvigstad, E., Moltzau, L. R., Sandnes, D., Birkeland, J. A. K., Sjaastad, I., Levy, F. O., 2006. The 5-HT4 receptor – a foetal gene reactivated in heart failure? J. Mol. Cell. Cardiol. 40, 944

Brattelid, T., Kvingedal, A. M., Krobert, K. A., Andressen, K. W., Bach, T., Hystad, M. E., Kaumann, A. J., Levy, F. O., 2004a. Cloning, pharmacological characterisation and tissue distribution of a novel 5-HT4 receptor splice variant, 5-HT4(i). Naunyn-Schmiedeberg's Arch. Pharmacol. 369, 616-628.

Brattelid, T., Qvigstad, E., Birkeland, J. A. K., Swift, F., Bekkevold, S. V. S., Krobert, K. A., Sejersted, O. M., Skomedal, T., Osnes, J. B., Levy, F. O., Sjaastad, I., 2007. Serotonin responsiveness through 5-HT2A and 5-HT4 receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle. J. Mol. Cell. Cardiol. 43, 767-779.

Brattelid, T., Qvigstad, E., Lynham, J. A., Molenaar, P., Aass, H., Geiran, O., Skomedal, T., Osnes, J.-B., Levy, F. O., Kaumann, A. J., 2004b. Functional serotonin 5-HT4 receptors in porcine and human ventricular myocardium with increased 5-HT4 mRNA in heart failure. Naunyn-Schmiedeberg's Arch. Pharmacol. 370, 157-166.

Chandra, M., Gupta, V., Johri, A. K., Misra, R., Kumar, A., Gujrati, V., Shanker, K., 1994. Serotonergic mechanisms in heart failure. Indian Heart J. 46, 153-156.

Chien, K. R., Knowlton, K. U., Zhu, H., Chien, S., 1991. Regulation of cardiac gene expression during myocardial growth and hypertrophy: molecular studies of an adaptive physiologic response. FASEB J. 5, 3037-3046.

Christ, T., Engel, A., Ravens, U., Kaumann, A. J., 2006. Cilostamide potentiates more the positive inotropic effects of (-)-adrenaline through b2-adrenoceptors than the effects of (-)-noradrenaline through b1-adrenoceptors in human atrial myocardium. Naunyn-Schmiedeberg's Arch. Pharmacol. 374, 249-253.

Claeysen, S., Sebben, M., Becamel, C., Bockaert, J., Dumuis, A., 1999. Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain. Mol. Pharmacol. 55, 910-920.

Cleland, J. G., 2003. Beta-blockers for heart failure: why, which, when, and where. Med. Clin. North Am. 87, 339-371.

Doughty, R. N., Whalley, G. A., Walsh, H. A., Gamble, G. D., Lopez-Sendon, J., Sharpe, N., 2004. Effects of carvedilol on left ventricular remodeling after acute myocardial infarction: the CAPRICORN Echo Substudy. Circulation 109, 201-206.

Dvorak, A. M., 1986. Mast-cell degranulation in human hearts. N. Engl. J. Med. 315, 969-970.

Fuder, H., Ries, P., Schwarz, P., 1994. Histamine and serotonin released from the rat perfused heart by compound 48/80 or by allergen challenge influence noradrenaline or acetylcholine exocytotic release. Fundam. Clin. Pharmacol. 8, 477-490.

Galindo-Tovar, A., Kaumann, A. J., 2008. Phosphodiesterase-4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (-)-adrenaline mediated through mouse cardiac b1-adrenoceptors. Br. J. Pharmacol. 153, 710-720.

Hara, M., Ono, K., Hwang, M. W., Iwasaki, A., Okada, M., Nakatani, K., Sasayama, S., Matsumori, A., 2002. Evidence for a role of mast cells in the evolution to congestive heart failure. J. Exp. Med. 195, 375-381.

Hasenfuss, G., Blanchard, E. M., Holubarsch, C., 1989. Influence of alpha- and beta-receptor stimulation on myocardial energetics in rabbit myocardium. Circulation 80 (suppl.II), 153.

Jahnel, U., Rupp, J., Ertl, R., Nawrath, H., 1992. Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle. Naunyn-Schmiedeberg's Arch. Pharmacol. 346, 482-485.

Kamel, R., Garcia, S., Lezoualc'h, F., Fischmeister, R., Muller, S., Hoebek, J., Eftekhari, P., 2007. Immunomodulation by maternal autoantibodies of the fetal serotoninergic 5-HT4 receptor and its consequences in early BALB/c mouse embryonic development. BMC. Dev. Biol. 7, 34.

Kaumann, A. J., 1994. Do human atrial 5-HT4 receptors mediate arrhythmias? Trends Pharmacol. Sci. 15, 451-455.

Kaumann AJ (2000) Gs Protein-Coupled Receptors in Human Heart. In: Kenakin T, Angus JA (eds) The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems. Springer-Verlag, Berlin Heidelberg New York, pp 73-116

Kaumann, A. J., Levy, F. O., 2006. 5-hydroxytryptamine receptors in the human cardiovascular system. Pharmacol. Ther. 111, 674-706.

Kaumann AJ, Sanders L (1998) 5-Hydroxytryptamine and human heart function: The role of 5-HT4 receptors. In: Eglen RM (ed) 5-HT4 receptors in the brain and periphery. Springer, Berlin, pp 127-148

Kaumann, A. J., Sanders, L., Brown, A. M., Murray, K. J., Brown, M. J., 1990. A 5-hydroxytryptamine receptor in human atrium. Br. J. Pharmacol. 100, 879-885.

Läer, S., Remmers, F., Scholz, H., Stein, B., Müller, F. U., Neumann, J., 1998. Receptor mechanisms involved in the 5-HT-induced inotropic action in the rat isolated atrium. Br. J. Pharmacol. 123, 1182-1188.

Lefebvre, H., Contesse, V., Delarue, C., Vaudry, H., Kuhn, J. M., 1998. Serotonergic regulation of adrenocortical function. Horm. Metab Res. 30, 398-403.

Lohse, M. J., Engelhardt, S., Eschenhagen, T., 2003. What is the role of beta-adrenergic signaling in heart failure? Circ. Res. 93, 896-906.

Lorrain, J., Grosset, A., O'Connor, S. E., 1992. 5-HT4 receptors, present in piglet atria and sensitive to SDZ 205-557, are absent in papillary muscle. Eur. J. Pharmacol. 229, 105-108.

Mongillo, M., McSorley, T., Evellin, S., Sood, A., Lissandron, V., Terrin, A., Huston, E., Hannawacker, A., Lohse, M. J., Pozzan, T., Houslay, M. D., Zaccolo, M., 2004. Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases. Circ. Res. 95, 67-75.

Nebigil, C. G., Choi, D. S., Dierich, A., Hickel, P., Le Meur, M., Messaddeq, N., Launay, J. M., Maroteaux, L., 2000. Serotonin 2B receptor is required for heart development. Proc. Natl. Acad. Sci. U. S. A 97, 9508-9513.

Nebigil, C. G., Jaffre, F., Messaddeq, N., Hickel, P., Monassier, L., Launay, J. M., Maroteaux, L., 2003. Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation 107, 3223-3229.

Omerovic, E., Bollano, E., Soussi, B., Waagstein, F., 2003. Selective b1-blockade attenuates post-infarct remodelling without improvement in myocardial energy metabolism and function in rats with heart failure. Eur. J. Heart Fail. 5, 725-732.

Osnes, J. B., Aass, H., Andersen, G. Ø., Skomedal, T., 2000. First-line antihypertensive therapy. Lancet 356, 509-510.

Poole-Wilson, P. A., 2003. ACE inhibitors and ARBs in chronic heart failure: the established, the expected, and the pragmatic. Med. Clin. North Am. 87, 373-389.

Qvigstad, E., Brattelid, T., Sjaastad, I., Andressen, K. W., Krobert, K. A., Birkeland, J. A., Sejersted, O. M., Kaumann, A. J., Skomedal, T., Osnes, J.-B., Levy, F. O., 2005a. Appearance of a ventricular 5-HT4 receptor-mediated inotropic response to serotonin in heart failure. Cardiovasc. Res. 65, 869-878.

Qvigstad E, Brattelid T, Sjaastad I, Molenaar P, Birkeland JA, Andressen KW, Krobert KA, Sejersted OM, Skomedal T, Osnes J-B, Kaumann AJ, Levy FO (2005b) Rationale for treatment of heart failure by blockade of ventricular serotonin receptors appearing in heart failure. Proceedings of the 25th European Section Meeting, International Society for Heart Research, Tromsø, Norway, June 21-25, 2005. Medimond International Proceedings, Bologna, Italy, pp 7-12

Qvigstad, E., Sjaastad, I., Brattelid, T., Nunn, C., Swift, F., Birkeland, J. A. K., Krobert, K. A., Andersen, G. Ø., Sejersted, O. M., Osnes, J. B., Levy, F. O., Skomedal, T., 2005c. Dual serotonergic regulation of ventricular contractile force through 5-HT2A and 5-HT4 receptors induced in the acute failing heart. Circ. Res. 97, 268-276.

Rahme, M. M., Cotter, B., Leistad, E., Wadhwa, M. K., Mohabir, R., Ford, A. P., Eglen, R. M., Feld, G. K., 1999. Electrophysiological and antiarrhythmic effects of the atrial selective 5-HT4 receptor antagonist RS-100302 in experimental atrial flutter and fibrillation. Circulation 100, 2010-2017.

Rajagopalan, S., Pitt, B., 2003. Aldosterone as a target in congestive heart failure. Med. Clin. North Am. 87, 441-457.

Rochais, F., Abi-Gerges, A., Horner, K., Lefebvre, F., Cooper, D. M., Conti, M., Fischmeister, R., Vandecasteele, G., 2006. A specific pattern of phosphodiesterases controls the cAMP signals generated by different Gs-coupled receptors in adult rat ventricular myocytes. Circ. Res. 98, 1081-1088.

Sawada, M., Ichinose, M., Ito, I., Maeno, T., McAdoo, D. J., 1984. Effects of 5-hydroxytryptamine on membrane potential, contractility, accumulation of cyclic AMP, and Ca2+ movements in anterior aorta and ventricle of Aplysia. J. Neurophysiol. 51, 361-374.

Saxena, P. R., Villalon, C. M., Dhasmana, K. M., Verdouw, P. D., 1992. 5-Hydroxytryptamine-induced increase in left ventricular dP/dtmax does not suggest the presence of ventricular 5-HT4 receptors in the pig. Naunyn-Schmiedeberg's Arch. Pharmacol. 346, 629-636.

Schoemaker, R. G., Du, X. Y., Bax, W. A., Bos, E., Saxena, P. R., 1993. 5-Hydroxytryptamine stimulates human isolated atrium but not ventricle. Eur. J. Pharmacol. 230, 103-105.

Schoemaker, R. G., Du, X. Y., Bax, W. A., Saxena, P. R., 1992. 5-Hydroxytryptamine increases contractile force in porcine right atrium but not in left ventricle. Naunyn-Schmiedeberg's Arch. Pharmacol. 346, 486-489.

Shah, A. M., Andries, L. J., Meulemans, A. L., Brutsaert, D. L., 1989. Endocardium modulates myocardial inotropic response to 5- hydroxytryptamine. Am. J. Physiol 257, H1790-H1797.

Skomedal, T., Borthne, K., Aass, H., Geiran, O., Osnes, J. B., 1997. Comparison between alpha-1 adrenoceptor-mediated and beta adrenoceptor-mediated inotropic components elicited by norepinephrine in failing human ventricular muscle. J. Pharmacol. Exp. Ther. 280, 721-729.

Sole, M. J., Shum, A., van Loon, G. R., 1979. Serotonin metabolism in the normal and failing hamster heart. Circ. Res. 45, 629-634.

Tendera, M., Ochala, A., 2001. Overview of the results of recent beta blocker trials. Curr. Opin. Cardiol. 16, 180-185.

The Xamoterol in Severe Heart Failure Study Group, 1990. Xamoterol in severe heart failure. Lancet 336, 1-6.

Vargas, M. L., Hernandez, J., Kaumann, A. J., 2006. Phosphodiesterase PDE3 blunts the positive inotropic and cyclic AMP enhancing effects of CGP12177 but not of noradrenaline in rat ventricle. Br. J. Pharmacol. 147, 158-163.

Vizir, V. A., Berezin, A. E., 2001. [Relationship between myocardial remodeling and neurohumoral activation in patients with cardiac failure]. Klin. Med. (Mosk) 79, 21-27.

Xiang, Y., Naro, F., Zoudilova, M., Jin, S. L., Conti, M., Kobilka, B., 2005. Phosphodiesterase 4D is required for b2 adrenoceptor subtype-specific signaling in cardiac myocytes. Proc. Natl. Acad. Sci. U. S. A. 102, 909-914.

Figure legend

Figure 1: Inotropic responses through adrenoceptors and serotonin receptors in cardiac ventricle and changes during heart failure. The figure illustrates how different signalling mechanisms activated through different G proteins result in different inotropic response patterns. The panels at the bottom illustrate single contraction/relaxation cycles before (continuous line) and after (dashed line) receptor stimulation. Note that cAMP-mediated signalling results in increased contractility with a shortening of the contraction-relaxation cycle, resulting from a combination of inotropic and lusitropic response, whereas cAMP-independent signalling (probably converging upon increased MLC phosphorylation) results in increased contractility without shortening of the contraction-relaxation cycle, i.e. without a lusitropic response. AC: adenylyl cyclase; AR: adrenoceptors; CC: sarcolemmal calcium channel; G: G protein; MLC: myosin light chain; MLCK: myosin light chain kinase; MLCP: myosin light chain phosphatase; PKA: cAMP-dependent protein kinase; PLB: phospholamban; PLC: phospholipase C; RhoK: Rho-associated protein kinase; RR: ryanodine receptor; TnI: troponin I

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