Cardiovascular Research

Cardiovascular Research 1998 40(3):516-522; doi:10.1016/S0008-6363(98)00198-9
© 1998 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Pino, R. Articles by Mugelli, A. Search for Related Content PubMed PubMed Citation Articles by Pino, R. Articles by Mugelli, A. Social Bookmarking          What's this?

Copyright © 1998, European Society of Cardiology

Effect of 5-HT₄ receptor stimulation on the pacemaker current I_f in human isolated atrial myocytes

Roberto Pino^a, Elisabetta Cerbai^a, Giancarlo Calamai^b, Franco Alajmo^b, Alberto Borgioli^b, Lucio Braconi^b, Massimo Cassai^b, Gian Franco Montesi^b and Alessandro Mugelli^a,*

^aDepartment of Pharmacology, University of Firenze, Viale G.B. Morgagni 65, 50134 Firenze, Italy
^bDepartment of Cardiosurgery, Azienda Ospedaliera Careggi, Firenze, Italy

^* Corresponding author. Tel.: +39-(55)-427-1264; fax: +39-(55)-427-1285; e-mail: mugelli@server1.pharm.unifi.it

Received 2 January 1998; accepted 22 April 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: 5-HT₄ receptors are present in human atrial cells and their stimulation has been implicated in the genesis of atrial arrhythmias including atrial fibrillation. An I_f-like current has been recorded in human atrial myocytes, where it is modulated by ß-adrenergic stimulation. In the present study, we investigated the effect of serotonin (5-hydroxytryptamine, 5-HT) on I_f electrophysiological properties, in order to get an insight into the possible contribution of I_f to the arrhythmogenic action of 5-HT in human atria. Methods: Human atrial myocytes were isolated by enzymatic digestion from samples of atrial appendage of patients undergoing corrective cardiac surgery. Patch-clamped cells were superfused with a modified Tyrode's solution in order to amplify I_f and reduce overlapping currents. Results and conclusions: A time-dependent, cesium-sensitive increasing inward current, that we had previously described having the electrophysiological properties of the pacemaker current I_f, was elicited by negative steps (–60 to –130 mV) from a holding potential of –40 mV. Boltzmann fit of control activation curves gave a midpoint (V_1/2) of –88.9±2.6 mV (n=14). 5-HT (1 µM) consistently caused a positive shift of V_1/2 of 11.0±2.0 mV (n=8, p<0.001) of the activation curve toward less negative potentials, thus increasing the amount of current activated by clamp steps near the physiological maximum diastolic potential of these cells. The effect was dose-dependent, the EC₅₀ being 0.14 µM. Maximum current amplitude was not changed by 5-HT. 5-HT did not increase I_f amplitude when the current was maximally activated by cAMP perfused into the cell. The selective 5-HT₄ antagonists, DAU 6285 (10 µM) and GR 125487 (1 µM), completely prevented the effect of 5-HT on I_f. The shift of V_1/2 caused by 1 µM 5-HT in the presence of DAU 6285 or GR 125487 was 0.3±1 mV (n=6) and 1.0±0.6 mV (n=5), respectively (p<0.01 versus 5-HT alone). The effect of 5-HT₄ receptor blockade was specific, since neither DAU 6285 nor GR 125487 prevented the effect of 1 µM isoprenaline on I_f. Thus, 5-HT₄ stimulation increases I_f in human atrial myocytes; this effect may contribute to the arrhythmogenic action of 5-HT in the human atrium.

KEYWORDS Human atrial myocytes; Serotonin (5-HT); 5-HT₄ receptor subtype; Arrhythmia (mechanisms); Pacemaker current

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Serotonin (5-hydroxytryptamine, 5-HT) exerts multiple effects on the cardiovascular system [1]. In the human and pig atrium, 5-HT causes positive chronotropic, inotropic, and lusitropic effects [2, 3], through stimulation of the 5-HT₄ receptor subtype. The human atrial 5-HT₄ receptor has been recently cloned; it appears to be different from the 5-HT₄ receptor isoform present in the brain and gastrointestinal tract [4]. Stimulation of the 5-HT₄ receptor is associated with the increase in cAMP levels and activation of cAMP-dependent protein kinase [2, 3, 5]. One of the consequences is the 5-HT₄ receptor-mediated increase of L-type calcium current (I_Ca,L) which has been demonstrated in isolated human atrial cardiomyocytes [6]. This effect is similar to that exerted by ß-adrenoceptor stimulation [6], and is the basis for the positive inotropic response to 5-HT. This positive inotropic effect is, however, observed in the human atrium, but not in the human ventricle [7]. Furthermore, the cardiac responses to 5-HT appear to be present only in few mammalian species (human, pig, monkey) and absent in common laboratory animals, such as the rat, guinea pig and rabbit [2, 6].

Due to its unique location in the human atrium, it has been suggested that 5-HT₄ receptors may be implicated in the control of atrial activity [8]. Since 5-HT has been reported to cause arrhythmias in the isolated human atrium [9], the suggestion that 5-HT released by platelets may be involved in the genesis of atrial arrhythmias and particularly of atrial fibrillation [8], appears particularly appealing. The mechanism for induction of the arrhythmia could be an intracellular calcium overload resulting in delayed afterdepolarizations and triggered activity, as consequence of the activation of the cAMP-dependent cascade induced by 5-HT through the stimulation of 5-HT₄ receptors and causing an increased calcium entry through L-type calcium channels [8].

We recently reported that the pacemaker current I_f is present in 82% of atrial myocytes isolated from the human right appendage of patients undergoing cardiac surgery [10]. The half maximal activation voltage (V_1/2) of I_f was around –86 mV which does not support a functional role for this current under physiological conditions in human atrial myocytes. In fact, the maximum diastolic potential in human atrial myocytes has been reported to be generally below this value (i.e., –70/–75 mV) [11–16], implying that the current undergoes little, if any, activation at these potentials. There are at least two ways by which such a current could become functionally important. First, its properties are modified by disease (e.g. atrial dilatation), similarly to what is observed for other ionic currents during hypertrophy or failure [17–22], or chronic atrial fibrillation [23], in such a way that it may become active under pathological conditions. A second possibility is that I_f activation curve is shifted toward less negative (and more physiological) potentials by the stimulation of relevant receptors. Recently, it was found that the V_1/2 of I_f in human atrial myocytes was significantly shifted toward less negative potentials by ß-adrenoceptor stimulation with isoprenaline [10]. In rabbit sino-atrial node cells it has been clearly demonstrated that the effect of cAMP on I_f is largely direct and does not involve the phosphorylation of the channel [24, 25]. It is likely that a similar mechanism is operative in the human heart; direct proof of the mechanism by which cAMP regulates I_f in the human atria is not available.

To get an insight into the possible contribution of I_f to the arrhythmogenic action of 5-HT in human atria, we thought that it would be important to know if 5-HT, through the stimulation of 5-HT₄ receptors, was able to affect I_f electrophysiological properties (amplitude and activation voltage), and if its effects on I_f were observed at the concentrations previously demonstrated to increase I_Ca,L.

Preliminary data have been presented in abstract form [26].

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell isolation and storage
The procedure used was similar to that described previously [10]. Small samples from human right atrial appendages were used to isolate single myocytes. The specimens, obtained from the tissue routinely excised during the preparation of extra-corporeal circulation, were brought to the laboratory in cold transport saline (see Section 2.4). In the laboratory the sample was placed in Ca²⁺-free oxygen-saturated solution. Epicardial fat and connective tissue were removed from the muscle specimen. The tissue samples were cut into small pieces of about 1 mm³ in size and gently stirred in a thermostated (35°C) Ca²⁺-free oxygen-saturated solution for 20 min in a tissue dissociation vessel [27, 28]. Cells were isolated by enzymatic dissociation in Ca²⁺-free solution plus collagenase type V (350 I.U. ml^–1, Sigma) and protease type XXIV (4 I.U. ml^–1, Sigma) for 20 min. After this step was completed, the samples were stirred for 20 min in a Ca²⁺-free solution containing collagenase type V (350 I.U. ml^–1). The solution was then replaced by a new enzymatic solution where the collagenase concentration was reduced to 175 I.U. ml^–1. During this final step, the supernatant was replaced by fresh collagenase solution every 15 min and the dissociation was followed by microscopic examination of the collected medium. Cells were then stored in a Kraft–Brühe solution and allowed to sediment under gravity for 15 min or with gentle centrifugation (5 min at 1000 rpm). The supernatant was removed and replaced by Ca²⁺-free Tyrode's solution. The Ca²⁺ concentration was raised stepwise (50, 100, 200, 500 µM). The isolated cells were kept at room temperature in Tyrode's solution containing penicillin (50 I.U. ml^–1) and streptomycin (50 I.U. ml^–1) and used within the day.

2.2 Patients
Tissue specimens were obtained from 18 patients of both sexes (age: 67±10 years, mean±S.D.) undergoing cardiac surgery, for cardiac vascular disease (67%) or aortic valve replacement (33%). All patients were in sinus rhythm; they were under treatment with various combinations of drugs, including Ca²⁺-antagonists, acetylsalicylic acid and nitrate compounds. Our previous results [10]suggested that the properties of I_f were not influenced by the underlying pathology or pharmacological treatment in any evident manner. However, patients chronically treated with ß-adrenergic blockers were excluded due to the reported sensitization of human atrial 5-HT₄ receptors induced by such therapy [29]. All patients gave their informed consent for the use of their tissue samples. The design of this study conforms to the Helsinki declaration [30]and was approved by the local ethical committee.

2.3 Electrophysiological recordings
The patch clamp technique (whole cell recording) was used to measure the electrophysiological properties of the isolated human atrial myocytes. Details of the equipment have been previously described [10]. Cells were placed in an experimental bath on the platform of an inverted microscope (Nikon Diaphot TMD, Japan). Experiments were performed using a patch amplifier (Axopatch-1B, Axon Instruments, CA, USA) interfaced to a personal computer by means of a general purpose DAC/ADC interface (Labmaster Tekmar, Scientific Solutions). Data were viewed on-line on an analogic oscilloscope and on the computer screen. Data acquisition and preliminary analysis were performed by means of the integrated software package PCLAMP (Axon Instruments). Cells were superfused with a normal Tyrode's solution (see Section 2.4); a modified Tyrode's solution was used during measurements of the hyperpolarization-activated inward current (I_f). Temperature was maintained in the range 36–37°C. Patch-clamp pipettes were prepared from glass capillary tubes (Garner, CA, USA) by means of a two-stage vertical puller (Hans Otchoski, Hamburg, Germany) and fire-polished just before use. Pipettes had a resistance of 1.5–2.5 M when filled with the internal solution.

For the experiments with drug application, the patch-clamped cell was superfused by means of a temperature-controlled micro-superfusor which allowed rapid changes of the solution bathing the cell; this enabled us to minimize current run-down and to reduce exposure of the other cells present in the experimental chamber to drugs. 5-HT was dissolved in distilled water to get a stock solution with a final concentration of 10 mM containing 1 mg/ml ascorbic acid. The stock solution was then diluted with Tyrode's solution to get the final 5-HT concentrations.

The selective 5-HT₄ antagonists, DAU 6285 (N-endo-8-methyl-8-azabicyclol[3.2.1]-oct-3-yl)-2-3-dihydro-2-oxo-1H-benzimidazole-1-carboxamide,hydrochloride) [31], and GR 125487 ([1-[2-[methylsulphonylamino]-ethyl]-4-piperidinyl]-methyl-1-methyl-1H-indole-3-carboxylate) [32, 33], were dissolved in distilled water to get the stock solutions (both 10 mM) and then diluted in Tyrode's solution up to the final concentrations. In the experiments aimed to test the role of cAMP in mediating 5-HT effect, the tip of the patch pipette was filled with the internal solution and then backfilled with the same solution also containing 200 µM cAMP (Sigma).

2.4 Solutions
The composition of the solutions employed was as follows (in mM): Transport saline: Lactic acid 286; NaCl 103; KCl 5.4; CaCl₂ 1.8; pH adjusted to 7.0 with NaOH. Isolation solution: NaCl 120, KCl 10, KH₂PO₄ 1.2, MgCl₂ 1.2, D-(+)-glucose 10, Hepes 10, taurine 20; pH adjusted to 7.2 with NaOH. Kraft-Brühe solution: KCl 85; K₂HPO₄ 30; MgSO₄ 5.0; glucose 20; pyruvic acid 5.0; creatine 5.0; taurine 5.0; EGTA 0.5; β-hydroxybutyric acid 5.0; succinic acid 5.0; K₂-ATP 2.0; pH adjusted to 7.0 with KOH. Normal Tyrode's solution: NaCl, 140; KCl, 5.4; CaCl₂, 1.0; MgCl₂, 1.2; Hepes–NaOH pH 7.35, 5.0; glucose, 10. Modified Tyrode's solution for hyperpolarization-activated inward current measurements: NaCl, 140; KCl 25; CaCl₂, 1.0; MgCl₂, 1.2; Hepes–NaOH pH 7.35, 5.0; glucose, 10; BaCl₂ 1.0 (to block inward rectifier-like channels); MnCl₂ 2.0 (to block I_Ca,L); 4-aminopyridine 0.5 (to block transient outward potassium current, I_to). Internal solution (used to fill the patch micro-pipettes): KCl 110, ATP/K⁺ 4.3, MgCl₂ 2.0, CaCl₂ 1.0, EGTA 11, Hepes/K⁺ 10, adjusted to pH 7.4 with KOH (free [Ca²⁺] <10^–8 M).

2.5 Data analysis and statistics
Data analysis and fitting were performed by using the ORIGIN 4.1 program (MicroCal, MA, USA) running on a Pentium personal computer. For fitting functions, non-linear models of convergence to solutions were used.

To evaluate steady-state values of the hyperpolarization-activated current, data were fitted to an exponential decay, which gave the time constant of I_f activation (). The difference between the extrapolated value and that of the current at the end of the hyperpolarization step was usually small (in the order of a few pA).

A Boltzmann model based on the partition theorem according to the general equation:

was fitted to the activation data, where V (mV) is the test membrane potential, V_1/2 (mV) is the fitted potential for half activation and k (mV) is related to the slope of the activation curve.

Cell membrane capacitance (C_m) was measured by applying a pulse of small amplitude (V=10 mV) starting from a holding potential of –70 mV. The current transient following this clamp protocol was fitted with a mono-exponential model to compute series resistance (R_s) and then C_m using the two equations given below:

and

where I_peak is the maximum level of current (relative to the holding current) following the depolarization and is the time constant of the exponential current decay. The membrane capacitance values obtained have been used to compute ionic current densities (I_f density in pA/pF).

Dose-effect curves were fitted by using the Hill equation:

where E_max is the maximum effect, k corresponds to the concentration at which 50% E_max is obtained (EC₅₀) and x is the concentration of drug.

Given the relatively small number of observations and the nature of the measured data that are likely to be non-normally distributed, non-parametric statistics (Wilcoxon) on paired data (one- or two-tail, according to the need) were used. All results are reported as means±S.E.M.; p<0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Cells isolated from the right atrial appendages of human hearts were relatively small, with an average membrane capacitance (C_m) of 62.1±3.6 pF (n=112). These data are consistent with the previously reported values for capacitance and geometrical dimensions of the cells [10]. As expected, following clamp steps to hyperpolarized membrane potentials, a time-dependent inward current was present in about 80% of the cells; since we have previously documented that this current has the electrophysiological properties of I_f [10], we will call it I_f.

Fig. 1 (panel A) shows the hyperpolarization-activated current (I_f) recorded from a human atrial myocyte bathed in the modified Tyrode's solution used to amplify I_f [10]. The current traces were evoked by steps at potentials in the range of the diastolic potential of normally polarized atrial myocytes. After superfusion with 1 µM 5-HT (panel B), the amplitude of the current at each potential was clearly increased. The activation curve obtained by plotting current amplitude vs. membrane potential was fitted by a Boltzmann function whose average midpoint was –88.9±2.6 mV (n=14), and the average current density, measured at –120 mV, was 4.3±2.0 pA/pF. Panel C of Fig. 1 shows that superfusion with 5-HT (1 µM) did not affect maximum current amplitude, but shifted the activation curve of I_f toward less negative potentials. The mean maximum current amplitude was 300±46 pA in controls (n=8) and 275±41 pA after 5-HT (n=8); also the slope factor k was not significantly changed being 10.4±1.2 mV in controls and 10.1±1.3 mV in the presence of 5-HT. The shift of the activation curve caused by 5-HT was 11.0±2.0 mV (n=8, p<0.001). In the presence of 5-HT the activation kinetics of I_f was consistently faster at every potential (Fig. 1, panel D).

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Effect of 5-HT on If in single human atrial myocytes. (A and B) Current traces recorded during steps to the potential indicated near each trace, in the absence (A) and presence (B) of 1 µM 5-HT. Holding potential=–40 mV; (C) corresponding I–V relationships for If, obtained by plotting If amplitude versus the step potential (=control; =1 µM 5-HT). Here and in the next figures, lines were obtained by fitting the experimental data points with a Boltzmann function (see Section 2). (D) Plot of time-constant reciprocals (1/) versus membrane potential (=control; =1 µM 5-HT).

 
Fig. 2 shows that the shift of the activation curve of I_f, measured with different concentrations of 5-HT, was dose-dependent. For comparison the dose–response curve of the effect of 5-HT on the shift of the voltage–activation relationship for I_f (present results) is shown together with that derived from the data regarding the effects of 5-HT on I_Ca,L amplitude [6]. It is apparent that the two curves are practically superimposable, the EC₅₀ for the effect on I_f being 0.14 µM, and that for the effect on I_Ca,L being 0.13 µM; in both cases the maximal effect of 5-HT was reached at the concentration of 1 µM. The increase in I_Ca,L caused by 5-HT in human atria involves an increase in intracellular cAMP [6].

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Concentration–effect curve for the shift of the I–V relationship of If (V1/2) by 5-HT. Closed circles represent mean±S.E.M. of values obtained in human atrial myocytes; each cell was exposed to a single 5-HT concentration. The number of cells tested is indicated in parenthesis. Open circles represent data derived from Fig. 1 of [6]. Lines were obtained by fitting data with a Hill equation, which gave an EC50 of 0.14 µM and 0.13 µM for the effect on If (solid line) and ICa,L (dashed line), respectively.

 
Fig. 3 suggests that a cAMP dependent pathway is also involved in the effect of 5-HT on I_f. In fact, increasing intracellular cAMP by adding it to the patch pipette at a concentration of 200 µM, causes a marked increase in I_f amplitude (panel A). Panel B shows the time-course of the cAMP effect: after rupturing the seal and dialysing the intracellular milieu with cAMP, I_f amplitude increases rapidly, reaching the steady-state in about 2 min (panel B). When the maximal effect is reached, i.e. after 3 min of cell dialysis, superfusion of the cell with a solution containing 5-HT (1 µM), does not cause any further increase in the current amplitude. This is clearly shown in panel A, where the current traces recorded before and after 5-HT are superimposable and in panel B which clearly demonstrates that superfusion with 5-HT (arrow) does not change the amplitude of I_f, which remains stable over time. Similar results were obtained in five cells. Panel C of Fig. 3 shows the current–voltage relationship obtained in the same myocyte before and after diffusion of cAMP into the cell, in the presence or in the absence of 5-HT. It is apparent that 5-HT does not exert any effect on the current–voltage curve when intracellular cAMP is increased. To demonstrate that the effect of 5-HT on I_f was due to the stimulation of the 5-HT₄ receptor subtype, cells were exposed to the selective 5-HT₄ antagonist DAU 6285 [34].

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Role of cAMP in mediating 5-HT effects on If. (A) current traces recorded during a step to –70 mV, immediately after rupturing the membrane under the patch pipette (), at the steady state, i.e., 3 min after seal rupture (, 3 min), and after addition of 1 µM 5-HT to the external solution (). (B) time-course of the effect of cAMP on If amplitude and lack of effect of 5-HT under this condition. Time 0 refers to the moment at which the seal was disrupted; arrow indicates the start of 5-HT superfusion. (C) Corresponding activation curves obtained before and after dialysing the cell with cAMP ( and , respectively) and in the presence of 5-HT ().

 
As shown in Fig. 4 (upper panels), exposure to DAU 6285 (10 µM) for 2 min completely prevented the effect of 5-HT on I_f amplitude. I_f-current traces recorded before and after application of 5-HT 1 µM (in the presence of DAU 6285) during steps at –60, –70, and –80 mV were identical. The activation curves obtained in control conditions, in the presence of DAU 6285 alone and of DAU 6285 plus 5-HT were fully superimposable. A similar effect was observed with the more selective 5-HT₄ antagonist GR 125487 [32, 33](data not shown).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 5-HT4 receptor blockade by DAU 6285 prevents the effect of 5-HT on If. (A and B): current traces recorded during steps to the potential indicated near each trace, in the absence (A) and presence (B) of 1 µM 5-HT, in a myocyte pretreated with 10 µM DAU 6285. Holding potential=–40 mV; (C) corresponding I–V relationships for If, obtained by plotting normalized If amplitude versus the step potential (=10 µM DAU 6285; =DAU 6285 plus 1 µM 5-HT; - - -=control curve).

 
Fig. 5 summarizes the results obtained with the two 5-HT₄ antagonists. Both DAU 6285 and GR 125487 completely prevented the shift of the I_f activation curve caused by 5-HT: in fact, the shift of V_1/2 caused by 1 µM 5-HT in the presence of 10 µM DAU 6285 or 1 µM GR 125487 was 0.3±1 mV (n=6) and 1.0±0.6 mV (n=5), respectively (p<0.01 versus 5-HT alone). The effect of 5-HT₄ receptor blockade was specific as demonstrated in Fig. 5. In fact, exposure of the cells to DAU 6285 did not prevent the effect of isoprenaline (ISO). A 1 µM concentration of ISO which increases I_f amplitude in human atrial myocytes [10], also increased I_f amplitude in the presence of the 5-HT₄ antagonist DAU 6285. Similar results were obtained with the 5-HT₄ antagonist GR 125487 (not shown).

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Specificity of 5-HT4 receptor blockade on the serotonin-induced shift of If activation curve (V1/2). (A) Summary of the effect of 5-HT on If in the absence and presence of the 5-HT4 selective antagonists, DAU 6285 or GR 125487. Each column represents the mean±S.E.M. of the shift in V1/2; the number of cells tested is indicated in parenthesis, *=p<0.05 versus 5-HT alone. (B, C) Traces recorded during hyperpolarizing steps to the potential indicated near each trace, in the absence (B) and presence (C) of 1 µM isoprenaline, in a myocyte pretreated with 10 µM DAU 6285. Holding potential =–40 mV.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present results demonstrate that the pacemaker current I_f in human atrial myocytes is modulated by 5-HT. 5-HT increases I_f amplitude through the stimulation of the 5-HT₄ receptor subtype and involves the activation of the cAMP pathway. The effect of 5-HT is completely prevented by the selective antagonists of the 5-HT₄ receptor subtype, GR 125487 and DAU 6285. These compounds have a selectivity at least 30-fold higher versus the 5-HT₄ receptor subtype compared to other 5-HT receptor subtypes [31, 32, 34]. Furthermore, it has been clearly demonstrated that the 5-HT₄ subtype is the only 5-HT receptor functionally present in the human atrium [4, 9]. The mechanism by which 5-HT₄ stimulation increase I_f amplitude is by shifting its activation voltage toward less negative values. The voltage of half maximal activation for I_f, i.e. the voltage at which the current is activated at half of its maximal value, in the human atrial cells is approximately –90 mV. This property makes unlikely a functional contribution of I_f under physiological conditions, since the maximum diastolic potential of human atrial cells is rarely so negative [11–16]. However, a shift of the activation voltage of I_f toward less negative values may completely change the scenario. Let us suppose that a shift occurs, as experimentally observed with 5-HT in our study, of 11 mV toward less negative values: this means that at –70 mV (a likely maximum diastolic potential for an atrial cell) a substantial fraction of the current (40%) will now be activated and possibly could play a functional role, i.e. leading to the development of a diastolic depolarization phase. The effect of 5-HT is similar to that previously observed with ß-adrenoceptor stimulation [10]. This is also confirmed in the present study; in fact when 5-HT₄ receptors are blocked, ISO causes an increase in I_f amplitude, which is indistinguishable from that caused by 5-HT. Thus, as for the effect of ß-adrenoceptor stimulation, it is likely that the effect occurs through the activation of the adenylyl cyclase pathway, leading to an increase in cAMP, that is the same pathway involved in the 5-HT₄ mediated increase in I_Ca,L [6]. This is strongly suggested also by the observed increase of I_f amplitude when the cAMP is diffused into the cell and by the lack of effect of 5-HT, when I_f is maximally activated by intracellular cAMP. However the mechanisms by which cAMP regulates I_Ca,L and I_f are likely to differ. In fact the effect of cAMP on I_f has been demonstrated to be largely direct and independent of channel phosphorylation [24, 25]. It is likely that such a mechanism could regulates I_f also in human atria, even if direct data are not available. It is worth noting that the EC₅₀ for the increase in I_Ca,L and I_f amplitude are similar ([6]and present results). The implications of these findings are quite interesting: a certain concentration of 5-HT able to stimulate the 5-HT₄ receptor, will cause a concomitant increase in both I_Ca,L and I_f in atrial cells. These two effects may cooperate in the arrhythmogenesis of the human atrium. In fact, as suggested by Kaumann [8], the increase in calcium current may lead to calcium overload, causing the transient inward current which underlies delayed afterdepolarizations (DADs) [35]. Epinephrine-induced DADs have been recorded in human atrial trabeculae [36]. DADs are a well known arrhythmogenic mechanism [37]; if their amplitude is sufficient they may reach the threshold giving rise to a spontaneous action potential (triggered activity). The increase in I_f amplitude may contribute to this phenomenon. In fact, if I_f is responsible for a diastolic depolarization, an increase in its amplitude can lead to a diastolic depolarization which, in its turn, may favor the attainment of threshold by a subthreshold DAD, thus causing an extrasystole, which may trigger the arrhythmia. In the presence of an arrhythmogenic substrate, such as a dilated atrium of an old patient [38], atrial fibrillation could develop.

Time for primary review 21 days.

	Acknowledgements

 
Supported in part by a grant from MURST (University of Firenze). We wish to thank Dr. Carla Ghelardini and Dr. Nicoletta Galeotti for useful discussion on 5-HT₄ receptor pharmacology.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

1. Villalon C.M., de Vries P., Saxena P.R. Serotonin receptors as cardiovascular targets. Drug Discovery Today (1997) 2:294–300.[CrossRef][Web of Science]
2. Kaumann A.J., Sanders L., Brown A.M., Murray K.J., Brown M.J. A 5-hydroxytryptamine receptor in human atrium. Br J Pharmacol (1990) 100:879–885.[Web of Science][Medline]
3. Kaumann A.J. 5-HT₄-like receptors in mammalian atria. J Neural Transm (1991) 34(suppl):195–201.[CrossRef]
4. Blondel O., Vandecasteele G., Gastineau M., et al. Molecular and functional characterization of a 5-HT₄ receptor cloned from human atrium. FEBS Lett (1997) 412:465–474.[CrossRef][Web of Science][Medline]
5. Sanders L., Kaumann A.J. A 5-HT₄-like receptor in human left atrium. Naunyn Schmiedebergs Arch Pharmacol (1992) 345:382–386.[Web of Science][Medline]
6. Ouadid H., Seguin J., Dumuis A., Bockaert J., Nargeot J. Serotonin increases calcium current in human atrial myocytes via the newly described 5-hydroxytryptamine-4 receptors. Mol Pharmacol (1992) 41:346–351.[Abstract]
7. Jahnel U., Rupp J., Ertl R., Nawrath H. Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle. Naunyn Schmiedebergs Arch Pharmacol (1992) 346:482–485.[Web of Science][Medline]
8. Kaumann A.J. Do human atrial 5-HT₄ receptors mediate arrhythmias? Trends Pharmacol Sci (1994) 15:451–455.[CrossRef][Medline]
9. Kaumann A.J., Sanders L. 5-Hydroxytryptamine causes rate-dependent arrhythmias through 5-HT₄ receptors in human atrium: facilitation by chronic β-adrenoceptor blockade. Naunyn Schmiedebergs Arch Pharmacol (1994) 349:331–337.[Web of Science][Medline]
10. Porciatti F., Pelzmann B., Cerbai E., et al. The pacemaker current I_f in single human atrial myocytes and the effect of β-adrenoceptor and A₁-adenosine receptor stimulation. Br J Pharmacol (1997) 122:963–969.[CrossRef][Web of Science][Medline]
11. Bush H.L., Gelband H., Hoffman B.F., Malm J.R. Electrophysiological basis for supraventricular arrhythmias. Arch Surg (1971) 103:620–624.[Abstract/Free Full Text]
12. Escande D., Loisance D., Planche C., Coraboeuf E. Age-related changes of action potential plateau shape in isolated human atrial fibers. Am J Physiol (1985) 249:H843–H850.[Web of Science][Medline]
13. Gelband H., Bush H.L., Rosen M.R., Myerburg R.J., Hoffman B.F. Electrophysiologic properties of isolated preparations of human atrial myocardium. Circ Res (1972) 30:293–300.[Abstract/Free Full Text]
14. Singer DH. Automaticity in human cardiac tissue. In: Rosen MR, Janse MJ, Wit AL, editors. Cardiac electrophysiology: a textbook. Mount Kisco, NY: Futura, 1990:247–263.
15. Ten Eick R.E., Singer D.H. Electrophysiological properties of diseased human atrium: Low diastolic potential and altered cellular response to potassium. Circ Res (1979) 44:545–557.[Free Full Text]
16. Wang Z., Fermini B., Nattel S. Delayed rectifier outward current and repolarization in human atrial myocytes. Circ Res (1993) 73:276–285.[Abstract/Free Full Text]
17. Beuckelmann D.J., Nabauer M., Erdmann E. Alterations of K⁺ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ Res (1993) 73:379–385.[Abstract/Free Full Text]
18. Cerbai E., Barbieri M., Mugelli A. Characterization of the hyperpolarization-activated current, I_f, in ventricular myocytes isolated from hypertensive rats. J Physiol (Lond) (1994) 481:585–591.[Abstract/Free Full Text]
19. Cerbai E., Barbieri M., Li Q., Mugelli A. Ionic basis of action potential prolongation of hypertrophied cardiac myocytes isolated from hypertensive rats of different ages. Cardiovasc Res (1994) 28:1180–1187.[Abstract/Free Full Text]
20. Cerbai E., Barbieri M., Mugelli A. Occurrence and properties of the hyperpolarization-activated current I_f in ventricular myocytes from normotensive and hypertensive rats during aging. Circulation (1996) 94:1674–1681.[Abstract/Free Full Text]
21. Cerbai E., Pino R., Porciatti F., et al. Characterization of the hyperpolarization-activated current, I_f, in ventricular myocytes from human failing heart. Circulation (1997) 95:568–571.[Abstract/Free Full Text]
22. Nuss H.B., Houser S.R. T-type Ca²⁺ current is expressed in hypertrophied adult feline left ventricular myocytes. Circ Res (1993) 73:777–782.[Abstract/Free Full Text]
23. Van Wagoner D., Pond A.L., McCarthy P.M., Trimmer J.S., Nerbonne J.M. Outward K⁺ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res (1997) 80:772–781.[Abstract/Free Full Text]
24. Accili E.A., Redaelli G., DiFrancesco D. Differential control of the hyperpolarization-activated current (i(f)) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes. J Physiol (Lond) (1997) 500:643–651.[Abstract/Free Full Text]
25. DiFrancesco D., Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature (1991) 351:145–147.[CrossRef][Medline]
26. Pino R., Cerbai E., Alajmo F., et al. Effect of 5-hydroxytryptamine (5-HT) on the pacemaker current I_f, in human atrial myocytes (Abstract). Circulation (1996) 94:2770.[Web of Science]
27. Jacobson SL, Altschuld RA, Hohl H. Muscle cell cultures from human heart. In: Piper HM, editor. Cell culture techniques in heart and vessel research. Springer, Berlin/Heidelberg/New York, 1990:75–98.
28. Pelzmann B., Schaffer P., Mächler H., Rigler B., Koidl B. Adenosine inhibits the L-type calcium current in human atrial myocytes. Naunyn Schmiedberg's Arch Pharmacol (1995) 351:293–297.[Web of Science][Medline]
29. Sanders L., Lynham J.A., Bond B., et al. Sensitization of human atrial 5-HT₄ receptors by chronic beta-blocker treatment. Circulation (1995) 92:2526–2539.[Abstract/Free Full Text]
30. World Medical Association Declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects. Cardiovasc Res 1997;35:2–3.
31. Schiavone A., Giraldo E., Giudici L., Turconi M., Sagrada A. DAU 6285: a novel antagonist at the putative 5-HT₄ receptor. Life Sci (1992) 51:583–592.[CrossRef][Web of Science][Medline]
32. Schiavi G.B., Brunet S., Rizzi C.A., Ladinsky H. Identification of serotonin 5-HT₄ recognition sites in the porcine caudate nucleus by radioligand binding. Neuropharmacology (1994) 33:543–549.[CrossRef][Web of Science][Medline]
33. Ghelardini C., Galeotti N., Casamenti F., et al. Central cholinergic antinociception induced by 5HT₄ agonists: BIMU 1 and BIMU 8. Life Sci (1996) 58:2297–2309.[CrossRef][Web of Science][Medline]
34. Dumuis A., Golzan H., Sebben M., et al. Characterization of a novel 5-HT₄ receptor antagonist of the azabicycloalkyl benzimidazolone class: DAU 6285. Naunyn Schmiedebergs Arch Pharmacol (1992) 345:264–269.[Web of Science][Medline]
35. Vassalle M., Mugelli A. An oscillatory current in sheep cardiac Purkinje fibers. Circ Res (1981) 48:618–631.[Abstract/Free Full Text]
36. Hou Z.Y., Lin C.I., Vassalle M., Chiang B.N., Cheng K.K. Role of acetylcholine in induction of repetitive activity in human atrial fibers. Am J Physiol (1989) 256:H74–H84.[Web of Science][Medline]
37. Wit AL, Rosen MR. Afterdepolarization ant triggered activity. In: Fozzard H, Heber E, Jennings R, editors. The heart and cardiovascular system. New York: Raven Press, 1986:1449–1491.
38. Narayan S.M., Cain M.E., Smith J.M. Atrial fibrillation. Lancet (1997) 350:943–950.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				S. Severi, C. Corsi, and E. Cerbai From in vivo plasma composition to in vitro cardiac electrophysiology and in silico virtual heart: the extracellular calcium enigma Phil Trans R Soc A, June 13, 2009; 367(1896): 2203 - 2223. [Abstract] [Full Text] [PDF]

				M. Liu, M. S. Geddis, Y. Wen, W. Setlik, and M. D. Gershon Expression and function of 5-HT4 receptors in the mouse enteric nervous system Am J Physiol Gastrointest Liver Physiol, December 1, 2005; 289(6): G1148 - G1163. [Abstract] [Full Text] [PDF]

				P. Bianchi, O. Kunduzova, E. Masini, C. Cambon, D. Bani, L. Raimondi, M.-H. Seguelas, S. Nistri, W. Colucci, N. Leducq, et al. Oxidative Stress by Monoamine Oxidase Mediates Receptor-Independent Cardiomyocyte Apoptosis by Serotonin and Postischemic Myocardial Injury Circulation, November 22, 2005; 112(21): 3297 - 3305. [Abstract] [Full Text] [PDF]

				D. I. Leftheriotis, G. N. Theodorakis, D. Poulis, P. G. Flevari, E. G. Livanis, E. K. Iliodromitis, A. Papalois, and D. Th. Kremastinos The effects of 5-HT4 receptor blockade and stimulation, during six hours of atrial fibrillation Europace, January 1, 2005; 7(6): 560 - 568. [Abstract] [Full Text] [PDF]

				G. Lonardo, E. Cerbai, S. Casini, G. Giunti, M. Bonacchi, F. Battaglia, B. Fiorani, P. L. Stefano, G. Sani, and A. Mugelli Atrial natriuretic peptide modulates the hyperpolarization-activated current (If) in human atrial myocytes Cardiovasc Res, August 15, 2004; 63(3): 528 - 536. [Abstract] [Full Text] [PDF]

				S. Barrere-Lemaire, C. Piot, F. Leclercq, J. Nargeot, and S. Richard Facilitation of L-type calcium currents by diastolic depolarization in cardiac cells: impairment in heart failure Cardiovasc Res, August 1, 2000; 47(2): 336 - 349. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Pino, R. Articles by Mugelli, A. Search for Related Content PubMed PubMed Citation Articles by Pino, R. Articles by Mugelli, A. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics