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266 Metoprolol induces cardiac beta-3 adrenergic receptor and Sphingosine 1 phosphate receptor 1 signals to prevent adverse Left-ventricle remodeling and dysfunction after myocardial infarction {#article-title-2} Background: β-adrenergic receptor (AR)-blockers are fore-front therapies against myocardial infarction (MI)-induced and other forms of heart failure (HF). Mechanisms accounting for these beneficial effects remain however only partially understood. In particular, due to the difference in receptor targeting and to the great variability in human HF-patients response, the specific mechanism of action of β-blockers is still under investigation. We have recently demonstrated, in an animal model of HF, that a reciprocal down-regulation occurs between β1AR and the cardioprotective sphingosine-1-phosphate (S1P) receptor-1 (S1PR1). Purpose: Hence, we hypothesize that, in addition to salutary actions due to direct β1AR blockade, agents such as metoprolol improve post-MI structural and functional outcome via restored protective S1PR1 signal, and we sought to determine mechanisms accounting for this effect. Methods and Results: In HEK293 cells and in in vitro cardiomyocytes, metoprolol (Meto) prevented isoproterenol (βAR agonist)-dependent S1PR1 down-regulation. Treatment of infarcted mice with Meto or S1P (one week after MI for 3 weeks) markedly ameliorated cardiac function and prevented remodeling, while preserving cardiac plasma membrane S1PR1 whose levels were down-regulated in untreated MI mice. Next, we co-infused infarcted mice with S1P and Meto, and found no additional beneficial effects. Since previous evidence attests that Meto can increase cardiac β3ARs levels and activity, and this receptor in adypocytes is responsible for S1P secretion, we measured basal and Meto-stimulated cardiac Sphingosine kinase 1 (SphK1), the enzyme responsible for S1P secretion, and circulating S1P levels in β3AR KO mice. These animals displayed markedly reduced levels of both, not rescued by Meto. Importantly, the β1AR blocker did not ameliorate post-MI dysfunction in β3AR KO mice. Conclusions: β1-blockers enhance β3AR-signaling, promoting the secretion of S1P that, in turn, activates the S1PR1 signalling. These signalling interactions represent a previously unrecognized mechanism whereby βAR blockers prevent post-MI decompensation and adverse remodelling. # 267 PDE8 is a novel regulator of cAMP signaling in human atrial fibrillation {#article-title-3} Purpose: Atrial fibrillation (AF) is associated with reduced L-type Ca2+ current (ICa,L) and altered cAMP-dependent signalling. Cyclic nucleotide phosphodiesterases (PDEs) degrade cAMP and regulate cAMP-mediated PKA-dependent phosphorylation of various proteins, including ICa,L channel subunits. PDE1-4 are the main PDE isozymes hydrolyzing cAMP in heart, but recent studies demonstrate the existence of a novel isoform PDE8 in ventricle. Here we assess the expression and localization of PDE8 in human atria of patients with sinus rhythm (SR), paroxysmal AF (pAF) and longstanding persistent (chronic) AF (cAF). Methods: mRNA (RT-qPCR) and protein (Western blot) levels of PDE8A and PDE8B isoforms were assessed in right atria of SR, pAF and cAF patients. Localization of PDE8A and PDE8B in human atrial cardiomyocytes was determined by immunofluorescence. Protein-protein interaction between ICa,L α1C channel subunit and PDE8B was studied by co-immunoprecipitation in the three rhythm groups. Results: PDE8 mRNA is present in human atrium and increases in both types of AF (Δct SR=0.84±0.03 n=15 vs pAF=1.03±0.03 n=8 and cAF =1.06±0.05 n=8, p

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.

180 Noninvasive ultrasound molecular imaging of the effect of atorvastatin on vascular inflammation {#article-title-2} Purpose: Non-invasive assessment of changes in vascular inflammatory activity may be of use in managing medical therapy in patients with atherosclerotic disease and for developing new candidate therapies. We hypothesized that molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression with contrast enhanced ultrasound (CEU) could be used to assess the effects of HMG-CoA reductase inhibitors on vascular inflammation. Methods: Mice deficient for the LDL-receptor and Apobec-1 editing protein that develop atherosclerosis in a time-dependent fashion were studied. Beginning at 12 weeks of age, mice received 8 weeks of either regular chow (n=10) or chow containing atorvastatin (0.01% wt/wt; n=12). At 20 weeks of age, CEU molecular imaging of the ascending aorta was performed after I.V. injection of VCAM-1-targeted (MBV) and control microbubbles (MBC).High frequency transthoracic ultrasound imaging (40MHz) was used for noninvasive assessment of plaque burden by measuring aortic wall thickness. Plasma levels of total cholesterol and LDL+VLDL cholesterol were measured. Histology with Movat's pentachrome was used to quantify plaque burden. Fluorescence immunohistology and Western blot was used to localize and quantify VCAM-1 expression in the aortic wall. Results: Atorvastatin treatment lowered plasma LDL+VLDL cholesterol levels by 20% in statin-treated animals vs control animals (189.1mg/dl vs 233 mg/dl, p=0.03). On histology, plaque burden was reduced by 61% in statin treated animals (3.7% of luminal area vs 9.2%, p

Among the five human 5-HT(4) (h5-HT(4)) receptor isoforms, the h5-HT(4(a)) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT(4) receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. Ligand binding or competition studies with two h5-HT(4) receptor agonists, serotonin and ML10302 and two h5-HT(4) receptor antagonists, [(3)H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. Ligand binding experiments revealed that [(3)H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT(4(a)) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [(3)H]-GR113808 and to serotonin. According to these results, we propose ligand-receptor complex models with serotonin and [(3)H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [(3)H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [(3)H]-GR113808.

RT - PCR technique was used to clone the human 5-HT(4(e)) receptor (h5-HT(4(e))) from heart atrium. We showed that this h5-HT(4(e)) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT(4(e)) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg(-1) protein, respectively. Expression of h5-HT(4(e)) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [(3)H]-GR113808 of a number of 5-HT(4) ligands, was consistent with that previously reported for other 5-HT(4) receptor isoforms. Surprisingly, we found that the rank order of potencies (EC(50)) of 5-HT(4) agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (K(i)) obtained from binding assays. Furthermore, EC(50) values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT(4(e)) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca(2+) currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT(4(e)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand and two 5-HT(4) antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT(4(e)) receptor has a pharmacological profile which is close to the native h5-HT(4) receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.

The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4PDE3PDE2PDE1.

1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (ICa), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazo-lo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the beta-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current I(K,ACh), in frog atrial and ventricular myocytes. 2. ODQ (10 microM) and LY 83583 (30 microM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 microM) on the isoprenaline (Iso)-stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 microM) had no effect under similar conditions. 3. In the absence of SNAP, LY 83583 (30 microM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 microM) or intracellular cyclic AMP (5-10 microM). ODQ (10 microM) had no effect under these conditions, while MBlue (30 microM) inhibited the Iso-stimulated ICa. 4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 microM acetylcholine (ACh) on ICa. 5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I(K,ACh) by ACh in the presence of intracellular GTP, and this inhibition was weakened when I(K,ACh) was activated by intracellular GTPgammaS. 6. The potentiating effect of LY 83583 on Iso-stimulated ICa was absent in the presence of either DL-dithiothreitol (DTT, 100 microM) or a combination of superoxide dismutase (150 u ml(-1)) and catalase (100 u ml(-1)). 7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of beta-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of M Blue.

Early studies in whole heart indicated that cGMP antagonized the positive inotropic effects of catecholamines and cAMP. Since the L-type Ca2+ channel current (ICa) plays a predominant role in the initiation and development of cardiac electrical and contractile activities, regulation of ICa by cGMP pathways has received much attention over the last ten years. Patch-clamp measurements of ICa in isolated cardiac myocytes reveal at least three different cGMP effectors that may participate to different degrees in different animal species and cardiac tissues in the regulation of ICa by cGMP. In frog ventricular myocytes, cGMP inhibits ICa by stimulation of a cGMP-stimulated cAMP phosphodiesterase (PDE2), whereas in rat ventricular myocytes, cGMP predominantly inhibits ICa via a mechanism involving activation of a cGMP-dependent protein kinase (cGMP-PK). In guinea pig, frog and human cardiomyocytes, cGMP can also stimulate ICa via an inhibition of a cGMP-inhibited cAMP phosphodiesterase (PDE3). This effect is most predominant in human atrial myocytes and appears readily during an activation of the soluble guanylate cyclase activity by low concentrations of nitric oxide (NO)-donors. Biochemical characterization of the endogenous phosphodiesterases and cGMP-PK in purified cardiac myocytes provide further evidence in support of these mechanisms of cGMP action on ICa. However, the regulation of cGMP levels by a variety of agents is not always consistent with their effects on contractility. In particular, the participation of cGMP and NO pathways in the regulation of cardiac ICa and contractility by acetylcholine is still questionable.

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.

1. The effects of the current (iex) generated by the Na-Ca exchange mechanism have been investigated on the electrical activity of a cardiac cell using the model of MC ALLISTER et al. (1975) for the Purkinje fibre action potential (AP). 2. The reversal potential and the steady-state value of iex were described by the same equations as for the squid axon (MULLINS, 1976). The maximal intensity and the time constant of iex were extrapolated from experiments using frog hearts. 3. A compact program written with the Adams method in Fortran IV (PLANT, 1979) allowed a minicomputer to be used. 4. The addition of iex to the previous model induced a prolongation of the AP and a reduction of the duration of the diastolic phase. 5. Increasing the maximal steady-state amplitude of iex may lead to early after depolarizations and oscillatory behaviour. These effects can be prevented by adequate changes in the amplitude of some potassium outward currents. 6. It is concluded that : (i) alterations of the Na-Ca exchange, e.g., by cellular Na loading, should induce variations of both AP repolarization and diastolic phase durations and, consequently, alter the beating rate ; (ii) iex could interfere with the outward currents whose characteristics should be reconsidered.

The effect of extracellular and intracellular application of forskolin on the voltage-sensitive calcium current, ICa, was studied in myocytes isolated from frog ventricle. Myocytes were isolated by enzymatic dissociation, and ICa was measured using the whole-cell configuration of the patch clamp technique modified to permit intracellular perfusion of various substances. Intracellular perfusion with forskolin (0.1 to 10 microM) had a negligible effect on ICa: ICa was increased 15 +/- 13% (mean +/- SE; N = 5). In contrast, superfusion of the cell with forskolin increased ICa significantly. The EC50 for the forskolin effect was 0.4 microM. A maximal 4.5-fold increase in ICa occurred with 3 microM forskolin. This is somewhat less than the maximal response to isoprenaline seen in this same series of experiments. The effects of forskolin, isoprenaline, and intracellular cAMP were not additive. In contrast, the effects of isoprenaline or intracellular cAMP and calcium channel agonists, such as Sandoz (+)202-791, were additive. This supports the hypothesis that the positive inotropic effects of forskolin are at least partly mediated by an increase in intracellular cAMP and a stimulation of ICa. The effects of forskolin were antagonized by acetylcholine (1 microM) or intracellular perfusion with cGMP. Acetylcholine on the average decreased forskolin-stimulated ICa 57 +/- 11% (N = 17). The relevance of these results to the suggestion that acetylcholine acts by mechanisms other than inhibition of adenylate cyclase is discussed.

The effects of extracellular application of cicletanine on the voltage-sensitive calcium current (ICa) was studied in isolated cells from frog ventricle. Myocytes were isolated by enzymatic dissociation and ICa was measured using the whole-cell configuration of the patch-clamp technique modified to permit intracellular perfusion with various substances. Cicletanine (10 to 100 microM) had no effect on control ICa. However, when ICa was enhanced by superfusion of the cell with saturating doses of beta-adrenergic agonist (isoprenaline, 2 microM) or by intracellular perfusion with maximal doses of cAMP (20 microM), cicletanine exerted a dual effect on ICa. At 10 microM, cicletanine generally induced a transient or sustained stimulation of ICa (5 to 40%), while 100 microM of the drug generally reduced ICa. The effects of cicletanine were reversible and not voltage-dependent. These results suggest that cicletanine affects ICa by acting on a mechanism occurring after cAMP synthesis, by enhancing cAMP concentration (e.g. through an inhibition of cAMP phosphodiesterase) or facilitating cAMP-dependent phosphorylation of the Ca channels.