Use-dependent blockade of cardiac pacemaker current (If) by cilobradine and zatebradine

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Abstract

The action of the bradycardiac agents, cilobradine (DK-AH269) and zatebradine (UL-FS49), on the cardiac pacemaker current (If) was investigated on short Purkinje fibres from sheep hearts, using the two-microelectrode voltage-clamp technique, and on isolated rabbit sino-atrial cells with the patch clamp technique. These drugs reduce dose dependently the amplitude of the If, without modifying either the voltage dependence or the kinetics of channel activation. When voltage-clamp pulse trains were applied, cilobradine induced a use-dependent blockade of If that was stronger and faster than that with zatebradine. Recovery from blockade during prolonged hyperpolarization was significantly faster with zatebradine. Presumably, both drugs block the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel by gaining access to a binding site within the open channel pore, and are removed from the blocking site by strong hyperpolarization with large inward If through the open channel. Cilobradine, compared to zatebradine blocks If more effectively and faster in both preparations. Consequently cilobradine strongly reduces the pacemaker diastolic depolarization rate and the cell's firing frequency.

Introduction

The molecular structure of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, responsible for the cardiac pacemaker current (If), has been recently elucidated. The general structure of the HCN channel seems to be closely related to the structure of the different voltage-gated K+ channels and the cyclic nucleotide-gated (CNG) channels. The α subunit of one monomer consists of six-transmembrane domains (S1 to S6), a pore loop between the S5 and S6 segments and a cyclic nucleotide-monophosphate (cNMP) binding domain in the C-terminal loop of the polypeptide (Kaupp and Seifert, 2001). Like the K+ channels, the HCN channel presumably consists of a tetramere, constructed by the association of four α subunits plus various β subunits Ulens and Tytgat, 2001, Yu et al., 2001. The exact composition of the wild HCN channel in both sino-atrial node and Purkinje cells is still not elucidated, because of the heterotetrameric structure of the α units and the presence of β subunits.

Bradycardiac agents are known to reduce the slope of the diastolic depolarization by blocking reversibly the If passing through the HCN channels Kobinger, 1989, Kobinger and Lillie, 1984, Van Bogaert and Goethals, 1987. The drug, ZD7288, selectively blocks the cardiac If in isolated sino-atrial node cells (BoSmith et al., 1993) and Purkinje fibres Berger et al., 1994, Berger et al., 1995. It has been shown in inside-out patches that ZD7288 blocks the mammalian HCN1 (mHCN1) channels by interacting with three amino acids located in the pore-lining S6 region after the open-channel configuration was induced by hyperpolarization (Ki Soon Shin et al., 2001). Although ZD7288 differs in some aspects regarding its mode of block of the HCN channels from what we know of zatebradine (UL-FS49) and cilobradine (DK-AH269) (see Section 4.1), this confirms our interpretation of the mechanism of If blockade by zatebradine in cardiac and neuronal HCN channels. The drug acts in the cationic form from the inside of the cell and can only gain access to the blocking binding site within the channel pore when the gate is open and the electrochemical gradient allows the entry of the positively charged drug into the channel. But at more negative voltages, with a large inward current crossing the open channel, the drug is swept from its binding site and relief from block is predominant Goethals et al., 1993, Raes et al., 1998, Van Bogaert, 1989, Van Bogaert et al., 1990, Van Bogaert and Goethals, 1992. The same mechanism of interaction between the HCN channel of sino-atrial node cells and the bradycardiac agent, ivabradine (S16257), has been proposed recently (Bucchi et al., 2002). The relevance of If in the control of heart rate makes it an important pharmacological target. A reduced heart rate improves the myocardial energetic balance, because heart rate is tightly coupled with oxygen consumption (Braunwald, 1971). Furthermore, prolongation of the diastole duration, when the sinus rate is lowered, increases the coronary blood flow (Kedem et al., 1990). If is one of the many currents involved in cardiac and neural pacemaker mechanisms DiFrancesco, 1993, Pape, 1996, Robinson and Siegelbaum, 2003. The bradycardiac agents, zatebradine and ivabradine Bois et al., 1996, Thollon et al., 1994, induce a limited heart rate reduction (30% of the control) in a rate-dependent manner (Kobinger and Lillie, 1984). Zatebradine, cilobradine, DK-AH3 and ivabradine never cause sinus arrest and have limited or no negative inotropic side effects Chen et al., 1992, Granetzy et al., 2000. Because of the presence of If in the retina (photoreceptors and ganglion cells) and in most neurons of both the central and peripheral nervous system Biel et al., 2002, Moosmang et al., 2001, Santoro and Tibbs, 1999, it is important to characterise the potency and selectivity of bradycardiac agents. It is known that zatebradine blocks If both in photoreceptors and dorsal root ganglion neurons Raes et al., 1998, Satoh and Yamada, 2002 at concentrations higher than those needed to block If in sino-atrial node cells. We now compare the If blockade by cilobradine, its stereoisomer, DK-AH268, and the racemic mixture, DK-AH3, with that by zatebradine, on both sheep Purkinje fibres and rabbit sino-atrial node cells. We conclude that cilobradine and its congeners are significantly more potent If blockers than zatebradine and all other bradycardiac agents. The mechanisms of this difference in potency are discussed.

Section snippets

Description of the drugs tested

DK-AH269.Cl (cilobradine) is a white crystalline powder, soluble in water. The molecular weight is 519.1. The chemical name is: (S)-(+)-1,3,4,5-Tetrahydro-7,8-dimethoxy-3-((1-(2-(3,4-dimethoxyphenyl)-ethyl)-3-piperidinyl)-methyl)-2H-3-benzazepin-2-one-hydrochloride (pKa=8.60, log P=1.17) (Fig. 1). The enantiomer of cilobradine is DK-AH268. The racemic mixture is DK-AH3.

UL-FS49.Cl (zatebradine) is a white crystalline powder, soluble in water. The molecular weight is 493.05. The chemical name is:

Action potentials (Fig. 2)

Under control conditions, there was a clear diastolic depolarization (phase 4) with a maximum diastolic depolarization rate (DDRmax) of 20.3 mV s−1. During continuous stimulation at 0.8 Hz in the presence of the drug, there was a progressive shift to a more negative maximal diastolic potential after the end of repolarization, with a gradual decrease of the maximal diastolic depolarization rate. As illustrated in Fig. 2, in the presence of 0.3 μM DK-AH3 the diastolic depolarisation was

If affecting drugs

If in cardiac tissues (Ih in nerve cells) is affected by a number of drugs. Some, like alinidine (Snyders and Van Bogaert, 1987) and ZD7288 (BoSmith et al., 1993), block the current without any use and frequency dependence (class 1). These drugs not only can presumably block the HCN channels in the closed state, at a holding potential of −30 mV, but are also active in cationic form from the inside on an open channel at mild hyperpolarization Harris and Constanti, 1995, Shin et al., 2001. Strong

Acknowledgements

This work was supported by the Born-Bunge Foundation (Cardiovascular section) and by Boehringer Ingelheim Pharma (Germany).

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