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(Circulation Research. 2005;97:268.)
© 2005 American Heart Association, Inc.

Integrative Physiology

Dual Serotonergic Regulation of Ventricular Contractile Force Through 5-HT_2A and 5-HT₄ Receptors Induced in the Acute Failing Heart

Eirik Qvigstad^*, Ivar Sjaastad^*, Trond Brattelid, Caroline Nunn, Fredrik Swift, Jon Arne Kro Birkeland, Kurt A. Krobert, Geir Øystein Andersen, Ole M. Sejersted, Jan-Bjørn Osnes, Finn Olav Levy, Tor Skomedal

From the Department of Pharmacology (E.Q., T.B., C.N., K.A.K., G.Ø.A., J.-B.O., F.O.L., T.S.), the Center for Heart Failure Research (E.Q., I.S., T.B., C.N., F.S., J.A.K.B., K.A.K., G.Ø.A., O.M.S., J.B.O., F.O.L., T.S.), and the Institute for Experimental Medical Research (I.S., F.S., J.A.K.B., O.M.S.), University of Oslo, Norway; and the Department of Cardiology (I.S., G.Ø.A.), Ullevaal University Hospital, Oslo, Norway.

Correspondence to Finn Olav Levy, Dept of Pharmacology, University of Oslo, PO Box 1057 Blindern, 0316 Oslo, Norway. E-mail f.o.levy{at}medisin.uio.no

	Abstract

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Cardiac responsiveness to neurohumoral stimulation is altered in congestive heart failure (CHF). In chronic CHF, the left ventricle has become sensitive to serotonin because of appearance of G_s-coupled 5-HT₄ receptors. Whether this also occurs in acute CHF is unknown. Serotonin responsiveness may develop gradually or represent an early response to the insult. Furthermore, serotonin receptor expression could vary with progression of the disease. Postinfarction CHF was induced in male Wistar rats by coronary artery ligation with nonligated sham-operated rats as control. Contractility was measured in left ventricular papillary muscles and mRNA quantified by real-time reverse-transcription PCR. Myosin light chain-2 phosphorylation was determined by charged gel electrophoresis and Western blotting. Ca²⁺ transients in CHF were measured in field stimulated fluo-4-loaded cardiomyocytes. A novel 5-HT_2A receptor-mediated inotropic response was detected in acute failing ventricle, accompanied by increased 5-HT_2A mRNA levels. Functionally, this receptor dominated over 5-HT₄ receptors that were also induced. The 5-HT_2A receptor-mediated inotropic response displayed a triphasic pattern, shaped by temporally different activation of Ca²⁺-calmodulin-dependent myosin light chain kinase, Rho-associated kinase and inhibitory protein kinase C, and was accompanied by increased myosin light chain-2 phosphorylation. Ca²⁺ transients were slightly decreased by 5-HT_2A stimulation. The acute failing rat ventricle is, thus, dually regulated by serotonin through G_q-coupled 5-HT_2A receptors and G_s-coupled 5-HT₄ receptors.

Key Words: serotonin • heart failure • 5-HT_2A receptor • Ca²⁺-calmodulin-dependent myosin light chain kinase • Rho-associated kinase

	Introduction

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Direct cardioexcitation by the neurotransmitter and vasoactive mediator serotonin (5-hydroxytryptamine [5-HT]) was believed until recently to be restricted to atria because of the lack of positive inotropic effects in nondiseased ventricular tissues from different species.^1,2 In contrast to previous reports, we recently discovered functional 5-HT₄ receptors and increased 5-HT₄ mRNA levels in chronic failing human and rat ventricle, demonstrating altered cardiac serotonin responsiveness in chronic heart failure.^3,4 Recent studies also demonstrated involvement of 5-HT_2B receptors in cardiac hypertrophy and failure, emphasizing the pathophysiological relevance of serotonin in cardiac disease.⁵

An extensive acute myocardial infarction (MI) causes a substantial loss of viable myocardium within a few hours and leads to progressive cardiac dysfunction. Consequently, a variety of compensatory mechanisms are initiated, such as increased neurohumoral drive, hypertrophy, and possibly reactivation of fetal genes, to rescue myocardial function.⁶ Heart failure is a progressive disease and gene expression, as well as phenotype, will be different at various stages. It is not known whether serotonin receptor expression increases gradually or is even higher in the acute phase. It is possible that the pattern of expression of various serotonin receptor subtypes is different at various stages of the disease.

We now report a composite inotropic serotonin response in failing left ventricle 3 days after MI, mediated predominantly through appearing 5-HT_2A receptors but also through appearing 5-HT₄ receptors. The 5-HT_2A response involved increased myosin light chain (MLC)-2 phosphorylation.

	Materials and Methods

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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

Congestive Heart Failure Model
Animal care was carried out according to the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). As described,⁷ an extensive MI was induced in 320 g male Wistar rats by coronary artery ligation. Three days later, rats were included, provided lung weight >2.0 g and clinical signs of overt congestive heart failure (CHF) were present. Left-ventricular end diastolic (LVEDP) and systolic (LVSP) pressures were measured by catheterization.⁷ Some rats were included at 4, 7, and 42 days postinfarction for estimation of pD₂ (–logEC₅₀) values for serotonin (Figure 1). Sham-operated animals (Sham) underwent similar surgery without coronary artery ligation.

View larger version (13K): [in this window] [in a new window]   Figure 1. Lower potency of serotonin in acute (3 to 4-day) than chronic (42-day) heart failure. Ordinate, pD2-values (–logEC50) for the serotonin-induced inotropic responses, measured as increase in (dF/dt)max.

Isolated Papillary Muscles
Posterior left ventricular papillary muscles were prepared and the contraction-relaxation cycles (CRCs) were recorded and analyzed as previously described.⁸ Maximal development of force (dF/dt)_max was used as an index of contractility, and inotropic responses to agonists were expressed by increases in (dF/dt)_max. Lusitropic responses were expressed as reduction in RT (time to 80% relaxation–time to peak force [TPF]). The descriptive parameters at the end of the equilibration period were used as control values. Blockers of adrenergic (1 µmol/L prazosin; 1 µmol/L timolol) and muscarinic cholinergic (1 µmol/L atropine) receptors were present in the experiments and, where indicated, the 5-HT_2A receptor blocker ketanserin (0.1 µmol/L) and the 5-HT₄ receptor blocker GR113808 (1.0 µmol/L). Serotonin was added to the organ baths cumulatively (concentration-response curves) or as a single bolus (10 µmol/L). The inhibition constant K_b of ketanserin was calculated from the ratio of EC₅₀ of serotonin in the absence and presence of ketanserin.

Quantitative Reverse-Transcription PCR
Left ventricular sample preparation, cDNA synthesis, and quantitative reverse-transcription PCR were performed as previously described.⁴

MLC-2 Phosphorylation
Phosphorylation of MLC-2 was determined by charged gel electrophoresis⁹ by using anti-ventricular MLC-2 monoclonal antibody, and the identity of the phosphorylated MLC-2 band was confirmed with a phospho-specific anti-MLC-2 (Ser19) antibody. MLC phosphorylation is reported as phosphorylated MLC-2 in percentage of total MLC-2.

Ca²⁺ Transients in Cardiomyocytes in Response to 5-HT_2A Stimulation
Ca²⁺ transients were measured at 37°C in field stimulated rat cardiomyocytes, enzymatically isolated, loaded with fluo-4 acetoxymethyl ester (AM) and cells were studied with a Zeiss LSM 510 laser scanning microscope as previously described.¹⁰ The cells were superfused with a solution containing (in mmol/L); 4 KCl, 1 MgCl₂, 145 NaCl₂, 10 HEPES, 10 glucose, 1.8 CaCl₂, 0.4 NaH₂PO₄ (pH 7.4).

Statistics
Data are expressed as mean±SEM from n animals. P<0.05 was considered statistically significant (Student t test and nonparametric Mann-Whitney test). When appropriate, Bonferroni corrections were made.

	Results

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Animal Characteristics
All CHF rats had large antero-lateral infarcts and signs of CHF including tachypnea, pleural effusion, and pulmonary congestion (160±11% increase in lung weight versus Sham at 3 days). Hemodynamic data at 3 days suggested diastolic and systolic heart failure with increased LVEDP (334±7%) and decreased LVSP (22±4.5%) versus Sham. CHF papillary muscles revealed 28±1.8% decreased basal contractile force versus Sham. Animal characteristics are summarized in Table 1.

View this table: [in this window] [in a new window]   Table 1. Animal Characteristics

Serotonin Potency Is Lower in Acute Than Chronic CHF
During the study of chronic CHF hearts,⁴ some hearts harvested 3, 4, and 7 days after MI displayed inotropic effects of serotonin with lower potency compared with chronic (42-day) CHF, pD₂ (–logEC₅₀) values ranging from 6.56±0.15 (n=5) at 3 days to 7.50±0.08 (n=6) at 42 days post-MI (Figure 1). This 10-fold lower sensitivity to serotonin could reflect a different 5-HT receptor population in acute than chronic CHF, where 5-HT₄ was the dominant receptor subtype.⁴ Accordingly, all further experiments were performed on acute (3-day) postinfarction failing hearts to systematically explore the possible contribution of different serotonin receptors to the inotropic response in acute CHF.

Both 5-HT_2A and 5-HT₄ Receptors Contribute to Inotropic Effects of Serotonin in Acute CHF
Serotonin (10 µmol/L) elicited a positive inotropic response in papillary muscles from acute CHF rats of 78.0±15.3% above control (n=7) compared with a negligible mechanical response in Sham hearts (2.2±1.0%, n=10, Figure 2A). The inotropic effect of serotonin in CHF was of similar magnitude as that of 10 µmol/L isoproterenol (76.8±10.2%, n=7), which was reduced compared with Sham (110.6±7.2%, n=10, P<0.05; Figure 2A). The lower sensitivity to serotonin in acute CHF (Figure 1) suggested involvement of a receptor with lower serotonin affinity, such as 5-HT_2A, also known to be present in rat atria,² possibly in combination with 5-HT₄, which we had found in chronic failing ventricle.⁴ With the 5-HT₄-selective antagonist GR113808 (1 µmol/L) and the 5-HT_2A-selective antagonist ketanserin (0.1 µmol/L) present, the positive inotropic response to 10 µmol/L of serotonin was essentially abolished (0.4±0.4%, n=8; Figure 2B), indicating that these 2 receptors fully accounted for the serotonin effect. The individual contribution of 5-HT_2A and 5-HT₄ to the serotonin response was determined using the selective antagonists. In the presence of GR113808 (1 µmol/L), serotonin (10 µmol/L) elicited a positive inotropic response of 44.9±6.6% (n=11; Figure 2B), completely reversible by ketanserin (0.1 µmol/L) to –1.3±2.1% (P<0.05; Figure 2C) when added at steady-state serotonin response. In the presence of ketanserin (0.1 µmol/L), serotonin (10 µmol/L) elicited a positive inotropic response of 20.2±9.2% (n=8; Figure 2B), completely reversible by GR113808 (1 µmol/L) to –3.4±1.5% (P<0.05, Figure 2D) when given subsequently to serotonin, similar to the previously demonstrated 5-HT₄ response in chronic CHF.⁴ Thus, acute failing hearts display a novel ventricular 5-HT_2A-mediated inotropic effect, in addition to a 5-HT₄-mediated inotropic effect.

View larger version (26K): [in this window] [in a new window]   Figure 2. The positive inotropic effect of serotonin in acute CHF is of similar size as that of isoproterenol and composed of 5-HT2A and 5-HT4-mediated components. A, Maximal inotropic response to serotonin (5-HT) (10 µmol/L) and isoproterenol (Iso) (10 µmol/L) in Sham and 3-day CHF papillary muscle. *P<0.05 Iso CHF vs Iso Sham. B, Maximal inotropic response to serotonin (5-HT) (10 µmol/L) in the absence or presence of GR113808 (GR) (1 µmol/L) and/or ketanserin (Ket) (0.1 µmol/L) as indicated. A and B, Inotropic responses are expressed as percentage increase in (dF/dt)max over control. C and D, Representative averaged CRCs in the presence of 1 µmol/L GR113808 (C) (5-HT2A component) or 0.1 µmol/L ketanserin (D) (5-HT4 component), before addition of agonist (Basal), at maximal steady state inotropic response to serotonin and after reversal with 0.1 µmol/L ketanserin (C) or 1 µmol/L GR113808 (D).

The possible different serotonin sensitivity of these 2 components was addressed by serotonin concentration-response curves in the presence of the respective blockers (Figure 3A). In the presence of GR113808 (1 µmol/L), the serotonin concentration-response curve exhibited a pD₂ value of 6.10±0.06 (n=7) and a maximal inotropic effect, at 10 µmol/L, of 24.3±5.4% above control. In contrast, the presence of ketanserin (0.1 µmol/L) revealed a pD₂ value of 7.15±0.01 (n=5) and a maximal inotropic effect, at 1 µmol/L of serotonin, of 14.6±4.6% above control. The absence of antagonists resulted in an intermediate pD₂ value of 6.56±0.15 (n=6) and a maximal inotropic effect, at 10 µmol/L of serotonin, of 22.4±4.6% above control. The 3 pD₂ values were significantly different from each other (P<0.05). The higher maximal inotropic effect in the single-dose (10 µmol/L serotonin) compared with the concentration-response experiments may reflect desensitization during cumulative agonist addition in the latter.

View larger version (31K): [in this window] [in a new window]   Figure 3. Comparison of 5-HT2A and 5-HT4-mediated inotropic responses in acute CHF. A and B, Concentration-response curves for serotonin in acute (3-day) CHF papillary muscles in the absence and presence of GR113808 (1 µmol/L) or ketanserin (0.1 µmol/L) (A) and in the presence of GR113808 (1 µmol/L), without and with 1.3 or 5 nmol/L ketanserin (B). Inotropic response is expressed in percentage of maximum in each experiment. All pD2 values were significantly different (P<0.05). C, Time course curves showing the development of the serotonin-mediated inotropic responses in acute CHF papillary muscles in the absence or presence of GR113808 (1 µmol/L) or ketanserin (0.1 µmol/L). Inset, responses normalized to illustrate the faster development of the 5-HT4 response.

5-HT_2A-Mediated Inotropic Response to Serotonin in Acute Failing Hearts
The affinity of ketanserin for the putative 5-HT_2A receptor was determined by constructing serotonin concentration-response curves in the presence of full 5-HT₄ blockade by GR113808 (1 µmol/L) without and with 1.3 nmol/L or 5 nmol/L ketanserin (Figure 3B). Parallel curves with pD₂ values of 6.10±0.06 (n=7), 5.70±0.01 (n=5), and 5.26±0.07 (n=7), respectively, with similar basal and maximum inotropic response (pooled average 20.2±2.7%, n=19) yielded an average inhibition constant (K_b) for ketanserin of 0.8 nmol/L (–logK_b=9.1), in agreement with reported ketanserin affinity at 5-HT_2A receptors.²

Qualitative Characteristics of Serotonin-Mediated Inotropic Effects in Acute CHF
5-HT_2A-Mediated Effects
In the presence of GR113808 (1 µmol/L), the time from serotonin addition to 50% and maximal 5-HT_2A-mediated inotropic response was 180±13 seconds and 6 to 7 minutes, respectively (Figure 3C). The time-response curve after 5-HT_2A stimulation followed a triphasic pattern with an initial fast component followed by a transient negative phase and subsequently a slowly developing sustained inotropic response (Figure 3C) similar to that described for ₁-adrenoceptors.¹¹ Serotonin did not significantly alter TPF and RT (Figure 2C, Table 2).

View this table: [in this window] [in a new window]   Table 2. Characteristics of Contraction-Relaxation Cycles in Sham and Acute CHF Before and After Subsequent Addition of Serotonin and Isoproterenol

5-HT₄-Mediated Effects
In the presence of ketanserin (0.1 µmol/L), the time from serotonin addition to 50% and maximal 5-HT₄ receptor-mediated inotropic response was 29±1.7 seconds and 1 to 2 minutes, respectively (Figure 3C). Serotonin now reduced TPF and RT significantly, demonstrating a lusitropic effect in addition to the inotropic effect, similar to that of ß-adrenoceptor stimulation (Figure 2D and Table 2).

Combined 5-HT_2A– and 5-HT₄-Mediated Effects
In the absence of GR113808 and ketanserin, the time from serotonin addition to 50% and maximal inotropic response was 42±10 seconds and 3 minutes, respectively (Figure 3C). Serotonin reduced TPF and RT slightly but not significantly in both the single bolus and concentration-response experiments (Table 2). This is different from the pure 5-HT₄ effect in this study, characterized by a hastening of relaxation, comparable to that of ß-adrenoceptor stimulation.⁸

Induction of 5-HT_2A and 5-HT₄ Receptor mRNA in 3-Day Postinfarction CHF
Real-time quantitative reverse-transcription PCR was used to measure changes in 5-HT_2A and 5-HT_4(b) mRNA levels normalized to GAPDH mRNA in CHF left ventricle compared with Sham (Figure 4). 5-HT_2A mRNA increased 3.8-fold (P<0.05) and 5-HT₄ mRNA increased 2.2-fold (P<0.05) in CHF compared with Sham, both in agreement with the functional data. Atrial natriuretic peptide (ANP) mRNA (CHF marker) was also significantly increased versus Sham.

View larger version (13K): [in this window] [in a new window]   Figure 4. Expression of 5-HT2A, 5-HT4(b), and ANP mRNA in Sham and acute CHF left ventricle. mRNA for 5-HT2A receptor (left), 5-HT4(b) receptor (middle), and ANP (right) was quantified by real-time quantitative reverse-transcription PCR in Sham and acute (3-day) CHF ventricle. The results were normalized to GAPDH and Sham was assigned a value of 1.

Molecular Mechanisms of the 5-HT_2A-Mediated Inotropic Response
Effects of MLC Kinase Inhibition
MLC kinase (MLCK) is involved in the ₁-adrenoceptor-mediated positive inotropic response in rat and human heart.¹² The 5-HT_2A-mediated mechanical response pattern in CHF resembles the ₁-adrenoceptor response in rat.¹¹ We investigated whether the 5-HT_2A response was dependent on the same signal transduction pathway. The MLCK inhibitor ML-9 (50 µmol/L) essentially abolished the sustained positive inotropic 5-HT_2A-mediated serotonin response (3.7±1.5%, n=6 with versus 44.9±6.6%, n=11 without, P<0.05; Figure 5A and 5B). The initial fast positive component was also inhibited by ML-9 (1.2±0.3% with versus 8.7±0.9% without, P<0.05; Figure 5A), whereas the transient negative component was enhanced (-8.4±2.5% with versus 2.5±1.5% without, P<0.05, Figure 5A). The ß-adrenoceptor-mediated response to isoproterenol was not inhibited by ML-9 (82.6±3.6%, n=6 with versus 76.8±10.2% without, n=7; not significant). ML-9 reduced basal contractility by 26.9±2.1% (n=6, P<0.05), consistent with previous results.¹²

View larger version (23K): [in this window] [in a new window]   Figure 5. Mechanisms of the ventricular 5-HT2A-mediated inotropic response in acute CHF. A, B, and D, Inotropic response to serotonin (10 µmol/L) in the presence of GR113808 (1 µmol/L) and with and without different inhibitors. A and B, Time course curves showing the development (A) and representative averaged (B) CRCs of the basal (Basal) and maximal serotonin-mediated (5-HT) inotropic responses in acute CHF in the absence or presence of inhibitors of MLCK (ML-9, 50 µmol/L), calmodulin (W-7, 50 µmol/L), ROCK (Y-27632, 50 µmol/L), or PKC (BIM, 10 µmol/L), as indicated. C, Phosphorylated MLC-2 as percentage of total MLC-2 in 3-day CHF papillary muscles. Papillary muscles from organ bath experiments, which were either untreated (control) or treated with serotonin (10 µmol/L) alone or in the presence of ML-9 (50 µmol/L), Y-27632 (50 µmol/L), or BIM (10 µmol/L), were freeze clamped in liquid N2. D, Development of the 5-HT2A-mediated inotropic response in the absence and presence of inhibitors of all PKC isoforms (BIM, 10 µmol/L), Ca2+-dependent PKC (,ß) isoforms (Gö6976, 10 µmol/L) and PKC (rottlerin, 5 µmol/L). Inset, Average initial positive (white bars) and transient negative (black bars) 5-HT2A-mediated inotropic response in the absence and presence of different PKC inhibitors.*P<0.05 vs no inhibitor.

Effects of Calmodulin Inhibition
The Ca²⁺-binding protein calmodulin activates MLCK and regulates smooth muscle contraction, but its involvement in myocardial G_q-coupled receptor-mediated inotropic effects has not been explored. The calmodulin antagonist W-7 (50 µmol/L) significantly inhibited the sustained positive inotropic 5-HT_2A effect (13.8±6.7%, n=6 with W-7 versus 44.9±6.6%, n=11 without, P<0.05; Figure 5A). The initial positive component was decreased (3.7±0.9% versus 8.7±0.9%, P<0.05; Figure 5A) and the transient negative component unaltered (Figure 5A). The ß-adrenoceptor-mediated response to isoproterenol was not significantly different from control (58.7±9.7, n=6 with W-7 versus 76.8±10.2%, n=7 without). W-7 decreased basal contractile force by 27.7±6.7%, n=6.

Effects of Rho-Associated Kinase Inhibition
Rho-associated kinase (ROCK) regulates MLC phosphorylation and the ₁-adrenoceptor-mediated response through both inactivation of myosin phosphatase and direct phosphorylation of MLC-2.^12,13 The selective ROCK inhibitor Y-27632 (50 µmol/L) significantly attenuated the sustained positive 5-HT_2A-mediated inotropic effect when added 45 minutes before 10 µmol/L of serotonin (17.7±4.9%, n=6 with versus 44.9±6.6%, n=11 without, P<0.05; Figure 5A and 5B). The initial positive and the transient negative components were not significantly changed by Y-27632 (Figure 5A). The ß-adrenoceptor-mediated response was not significantly affected by Y-27632 (86.9±3.7%, n=6, with versus 76.8±10.2%, n=7 without). Y-27632 decreased basal contractile force by 32.0±2.2%, n=6. When Y-27632 was added after 5-HT_2A stimulation, the response was reversed from 42.0±9.8% to –12.1±5.5% (n=5, P<0.05). This corresponds to 78% reversal of the inotropic 5-HT_2A response when corrected for the cardiodepressive effect of Y-27632.

Effects of Protein Kinase C Inhibition
The negative inotropic component of the ₁-adrenoceptor response in rat and mouse heart is mediated through the diacylglycerol-protein kinase C (PKC) signal transduction pathway.^14,15 A 5-HT_2A-mediated negative inotropic component has not been previously demonstrated. Bisindolylmaleimide I (BIM) (10 µmol/L), an inhibitor of all PKC isoforms, completely abolished the transient negative 5-HT_2A response (Figure 5A), yielding an almost monophasic time course (Figure 5A). In addition, the initial and the sustained positive inotropic responses were increased to 17.6±1.6% (n=5, versus 8.7±0.9% without BIM, n=11, P<0.05) and 50.6±4.7% (n=5 versus 44.9±6.6%, n=11 without BIM, not significant; Figure 5A), respectively. BIM increased basal contractility by 11.5±3.1%. Gö6976 (10 µmol/L), a selective inhibitor of Ca²⁺-dependent PKC (,ß) isoforms,¹⁶ did not significantly change the triphasic 5-HT_2A response (Figure 5D), whereas the relatively selective PKC inhibitor rottlerin¹⁷ (1, 3, and 5 µmol/L) demonstrated a concentration-dependent inhibition of the transient negative 5-HT_2A component (Figure 5D), possibly implicating PKC in this negative component.

MLC-2 Phosphorylation Levels in Papillary Muscles After 5-HT_2A Stimulation
In the basal state, phosphorylated MLC-2 (all values given as percentage of total MLC-2) was significantly lower in CHF (14.3±2.7%, n=3; Figure 5C) compared with Sham (23.8±1.1%, n=3, P=0.03). Serotonin (10 µmol/L; 1 µmol/L GR113808 present) led to a 1.79-fold increase in phosphorylated MLC-2 (25.6±2.0%, n=4, P=0.02) that was abolished when muscles were treated with inhibitors of MLCK (ML-9; 16.0±0.3%, n=3) or ROCK (Y-27632; 15.8±1.1%, n=4) but not with the PKC inhibitor BIM (25.9±4.7%, n=4).

5-HT_2A-Mediated Effects on Ca²⁺ Transients in Isolated Cardiomyocytes
The 5-HT_2A effects on Ca²⁺ transients in CHF were measured with a laser scanning confocal microscope in field-stimulated cardiomyocytes loaded with fluo-4. Figure 6 shows representative line scan diagrams illustrating Ca²⁺ transients recorded from CHF myocytes before and during selective 5-HT_2A stimulation with serotonin (10 µmol/L; 1 µmol/L GR113808 present). The 5-HT_2A stimulation data were corrected for a 6.3% decrease in maximal systolic fluorescence normalized to diastolic fluorescence (F/F₀) over time in nonstimulated myocytes (not shown). 5-HT_2A stimulation induced an 8.9% reduction in the Ca²⁺ transients (P<0.05). As a control, isoproterenol (10 µmol/L) induced a >50% increase in the Ca²⁺ transient.

View larger version (16K): [in this window] [in a new window]   Figure 6. Line scan tracings and summarized Ca2+ transients in basal and after 5-HT2A stimulation. A (left), Representative line scan diagrams recorded from CHF myocytes loaded with fluo-4 in the presence (5-HT2A) and absence (Basal) of serotonin (10 µmol/L). A (right), Spatially averaged Ca2+ transients derived from the line scan diagrams to the left. B, The mean Ca2+ transients were 8.9% lower following 5-HT2A stimulation (right column) compared with basal (left column) (P<0.05). C, Isoproterenol (Iso) (10 µmol/L) induced a significant increase in the Ca2+ transients compared with basal in control experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We demonstrate, for the first time, induction of functional 5-HT_2A serotonin receptors in acute failing cardiac ventricle, accompanied by increased 5-HT_2A mRNA expression. Stimulation of G_q-coupled 5-HT_2A receptors elicits a triphasic inotropic response, resulting from temporally distinct activation of Ca²⁺-calmodulin-dependent MLCK, ROCK, and inhibitory PKC and increases MLC-2 phosphorylation. 5-HT_2A stimulation did not increase Ca²⁺ transients. We also found induction in acute CHF of functional 5-HT₄ receptors and 5-HT₄ mRNA, previously documented in chronic CHF.^3,4 Quantitatively, the 5-HT_2A response dominates over the 5-HT₄ response in acute failing ventricle.

Two independent pharmacological criteria identify the functional non-5-HT₄ receptor in acute CHF as 5-HT_2A. First, a hallmark of 5-HT_2A receptors is low affinity for serotonin, translating into low potency in functional assays. The GR113808-resistant serotonin response showed the same potency as that of 5-HT_2A in rat atria,² 10-fold lower than the potency of the ketanserin-resistant response (5-HT₄; Figure 3A). Second, the high affinity for the 5-HT_2A-selective antagonist ketanserin (pK_b=9.1), as determined by the shifts of the serotonin concentration-response curves, is only compatible with the 5-HT_2A. For example, with a pK_i of 7.3 and 5.5 for rat 5-HT_2C¹⁸ and 5-HT_2B receptors,¹⁹ respectively, the ketanserin concentrations (1.3 nmol/L and 5.0 nmol/L) used here would not move the serotonin concentration-response curves for 5-HT_2B or 5-HT_2C-mediated responses. Similarly, the presence of 0.1 µmol/L ketanserin would cause <1% and 10% decreases in a 5-HT_2B and 5-HT_2C response, respectively. Thus, among 5-HT receptors, only 5-HT_2A and 5-HT₄ receptors regulate contractility in the acute failing ventricle.

Stimulation of G_q-coupled 5-HT_2A receptors in acute failing ventricle elicits a rather complex temporally regulated inotropic response, different from that of G_s-coupled 5-HT₄ receptors. Composed of an initial positive, a transient negative and a sustained positive phase, the response resembles that of the ₁-adrenoceptor stimulation in rat.¹¹ In contrast to cAMP-dependent effects on cardiac contractility (eg, 5-HT₄, ß-adrenoceptor), the 5-HT_2A response develops slowly (Figure 3) and is characterized by a symmetrical change in the CRC with unchanged or slightly prolonged TPF and RT (Figure 2C and Table 2), qualitative characteristics typical of cardiac G_q-coupled receptors (eg, ₁-adrenoceptors and endothelin receptors). Despite a robust positive inotropic response in acute failing hearts, we observed no increase in Ca²⁺ transients after 5-HT_2A stimulation, in contrast to a >50% increase after activation of ß-adrenoceptors. Accordingly, the 5-HT_2A-mediated positive inotropic response is probably attributable to an increased myofilament Ca²⁺ sensitivity in acute failing ventricle.

In smooth muscle, where phosphorylation of MLC-2 is necessary for force production, G-protein-coupled receptor-mediated MLCK activation and MLC phosphatase inhibition determine the MLC-2 phosphorylation state and, thus, contraction.²⁰ In cardiac muscle, MLC-2 phosphorylation has been shown to be important for regulation of contraction by increasing myofibrillar Ca²⁺ sensitivity.²¹ Furthermore, regulation of cardiac contractility through G_q-coupled receptors, eg, ₁-adrenoceptor and endothelin receptors, is mediated through a Ca²⁺-sensitizing mechanism assumed to involve increased MLC-2 phosphorylation.^12,22,23 On this background and given the similarity of 5-HT_2A and ₁-adrenoceptor inotropic responses, we wanted to determine possible involvement of MLC-2 phosphorylation.

The specific MLCK inhibitor ML-9²⁴ selectively inhibits cAMP-independent inotropic effects (eg, of phenylephrine, endothelin) in heart tissue,¹² indicating that these effects are mediated via increased MLC-2 phosphorylation. Similarly, the novel ventricular 5-HT_2A-mediated inotropic response was almost abolished by ML-9. Furthermore, the 5-HT_2A-mediated increased phosphorylation of MLC-2 at Ser19 was also inhibited by ML-9. We found no inhibition of the ß-adrenoceptor-mediated effect by ML-9, nor was the 5-HT₄-mediated inotropic effect inhibited by ML-9 (not shown). Accordingly, we suggest that the sustained positive inotropic response to serotonin through 5-HT_2A receptors in acute CHF depends on MLCK-mediated phosphorylation of MLC-2. This was further supported by the observed attenuation of the positive 5-HT_2A response by inhibition of calmodulin, which is involved, for example, in activation of MLCK (Figure 5A). The present study is the first to demonstrate a 5-HT_2A-mediated increase in MLC-2 phosphorylation at Ser19 in failing ventricular tissue. The decrease in basal MLC-2 phosphorylation in CHF compared with Sham is in accordance with a previous study.²⁵

In smooth muscle, the Rho/ROCK pathway contributes to agonist-mediated contraction mainly through inhibition of myosin phosphatase, thus increasing phosphorylated MLC-2.²⁶ ROCK is activated by G_q-coupled receptor signaling via the small G-protein RhoA.²⁷ The pyridine derivative, Y-27632, is a selective ROCK inhibitor,²⁶ and there is now increasing evidence that the Rho/ROCK pathway is involved in agonist (eg, phenylephrine)-induced inotropic effects.^12,28 In the present study, Y-27632 inhibited the 5-HT_2A-mediated positive inotropic effect both when added before and at steady-state response to serotonin and abolished the increased MLC-2 phosphorylation, suggesting involvement of the Rho/ROCK pathway in both increased MLC-2 phosphorylation and positive inotropic response through the novel ventricular 5-HT_2A receptor. In contrast, Y-27632 did not inhibit the cAMP-dependent ß-adrenoceptor-mediated inotropic response, consistent with previous reports.^12,29 Both ML-9 and Y-27632 decreased basal contractility in failing ventricular tissue, demonstrating an importance of MLCK and ROCK in the maintenance of basal cardiac contractility.

PKC, activated, for example, by G_q-coupled receptors through phospholipase Cß activation, plays a pivotal role in smooth muscle contraction by regulating the Ca²⁺ sensitivity through MLC-2 phosphorylation.³⁰ In the present study, PKC inhibition by BIM did not inhibit the positive inotropic 5-HT_2A response or alter the phosphorylation state of MLC-2 after serotonin stimulation. In contrast, BIM inhibited the negative inotropic component of the 5-HT_2A response, which is in agreement with reported involvement of PKC in ₁-adrenoceptor-mediated negative inotropic effects in mouse and rat heart.^15,31 BIM, an isoform-nonselective PKC inhibitor³² produced a moderate increase in basal contractile force, probably reflecting the presence of a constitutive PKC activity suppressing contractility in failing ventricular tissue, consistent with previous reports.³³ Whereas Gö6976 (10 µmol/L) which selectively inhibits the Ca²⁺-dependent PKC isoenzymes (PKC: IC₅₀=2.3 nmol/L; PKCß_I: IC₅₀=6.2 nmol/L) without affecting the kinase activity of Ca²⁺-independent isoforms (PKC, , , µ)¹⁶ did not influence the triphasic 5-HT_2A response, rottlerin (1, 3, and 5 µmol/L) dose dependently inhibited the transient negative inotropic component without affecting the positive components. The PKC inhibitor rottlerin can, to some extent, differentiate between different isoforms with at least 10-fold selectivity for PKC (IC₅₀3 µmol/L) compared with other isoforms (PKC, ß, , µ: IC₅₀30 µmol/L; PKC, , : IC₅₀ 80 to 100 µmol/L).¹⁷ Taken together, this suggests a possible involvement of PKC in the transient negative 5-HT_2A response, in agreement with the observed PKC-dependent ₁-adrenergic negative inotropic response in mice.¹⁵

We did not observe an involvement of the mitogen-activated protein kinase (MAPK) pathways in the 5-HT_2A-mediated positive inotropic response. The presence of the p38 MAPK inhibitor (SB203580, 10µmo/L) or the ERK cascade inhibitor (PD98059, 10 µmol/L) did not affect the 5-HT_2A-mediated inotropic response (data not shown). These results are in agreement with previous reports demonstrating no involvement of MAPK pathways on ₁-adrenoceptor-mediated inotropic effects.¹²

In both acute and chronic failing ventricle, serotonin alone elicited an inotropic response of comparable size to the attenuated ß-adrenoceptor-mediated effect. This emphasizes the dramatic alterations of cardiac serotonin responsiveness and the ability to modulate cardiac contractility after MI and CHF. Induction of functional 5-HT₄ and 5-HT_2A receptors in the acute failing postinfarction ventricle may be considered a compensatory mechanism to rescue contractile function after massive loss of viable myocardium. Whether sufficient levels of endogenous serotonin are present to activate serotonin receptors in the heart is currently unknown. In cardiac tissue serotonin has been identified in vascular beds, mast cells, and sympathetic nerve endings,^34,35 and increased plasma levels of serotonin in CHF and hypertensive disease and after MI have been reported.^36–38

In conclusion, the acute failing rat ventricle is dually regulated by serotonin through cAMP-independent mechanisms via G_q-coupled 5-HT_2A receptors and cAMP-dependent mechanisms via G_s-coupled 5-HT₄ receptors analogous to what is observed for the adrenoceptors (₁ and ß). The mechanism of the 5-HT_2A-mediated inotropic response involves phosphorylation of MLC-2 at Ser19 similar to stimulation of ₁-adrenoceptors. A further challenge is to clarify the importance of the myocardial serotonergic system and the conditions and signals evoking these changes in the failing heart.

	Acknowledgments

 
Supported by The Norwegian Council on Cardiovascular Diseases, The Research Council of Norway, Anders Jahre’s Foundation for the Promotion of Science, The Novo Nordisk Foundation, and The Family Blix foundation. Line Solberg and Nils Tovsrud provided technical assistance.

	Footnotes

 
*Both authors contributed equally to this work.

Original received February 23, 2005; revision received June 13, 2005; accepted June 29, 2005.

	References

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