Patrick Bois

Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells

1 The effect of the bradycardic agent S 16257 on the main ionic mechanisms of diastolic depolarization in sinoatrial node cells isolated from rabbit heart, was investigated by the patch‐clamp technique in whole‐cell and macro‐patch recordings. 2 In whole‐cell conditions, S 16257 induced a marked exponential use‐dependent blockade of the hyperpolarization‐activated If current, without shift of the voltage range of its activation curve. The rate of block increased with the drug concentration. The IC50 for the block of If was 2.8 × 10−6 m. 3 A similar use‐dependent decline of If was obtained with 3 μm S 16257, in cell‐attached and in inside‐out macro‐patch configurations, suggesting that the bradycardic agent interacts with If channels from the inside of the cell. 4 A high concentration of S 16257 (10 μm) had no detectable effect on T‐type calcium current and slightly decreased L‐type calcium current (−18.12±0.66%), without significant use‐dependent blockade. 5 S 16257 had no effect on the delayed outward potassium current IK at 3 μm and slightly decreased it only at high concentrations, −16.3±1.2% at 10 μm. In contrast, zatebradine, another bradycardic agent, reduced IK by 20.3±2.5% at 3 μm. 6 In conclusion, S 16257 may lower heart rate without significant negative inotropic action. In comparison with zatebradine, S 16257 had less effect on IK suggesting less prolongation of repolarization time.

Functional characterization of a Ca2+‐activated non‐selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i‐sensitive currents: the Na+–Ca2+ exchange current, a Ca2+‐activated chloride current and a Ca2+‐activated non‐selective cationic current. Using the cell‐free configuration of the patch‐clamp technique, we have characterized the properties of a Ca2+‐activated non‐selective cation channel (NSCCa) in freshly dissociated human atrial cardiomyocytes. In excised inside‐out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channels open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 μm. Channel activity was reduced in the presence of 0.5 mm ATP or 10 μm glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 mm flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the ‘TRP melastatin’ subfamily. In conclusion, the NSCCa channel is a serious candidate to support the delayed after‐depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

9‐Phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels

Background and purpose: TRPM4 and TRPM5 are calcium‐activated non‐selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5.

Mechanism of muscarinic control of the high-threshold calcium current in rabbit sino-atrial node myocytes

The mechanism of the action of acetylcholine (ACh) on the L-type calcium current (ICa,L) was examined using a whole-cell voltage-clamp technique in single sino-atrial myocytes from the rabbit heart. ACh depressed basal ICa,L at concentrations in the range 0.05–10 μM, without previous β-adrenergic stimulation. The ACh-induced reduction of ICa,L was reversed by addition of atropine, indicating that muscarinic receptors mediate it. Incubation of cells with a solution containing pertussis toxin led to abolition of the ACh effect, suggesting that this effect is mediated by G proteins activated by muscarinic receptors. Dialysis of cells with protein kinase inhibitor or 5′-adenylyl imidodiphosphate, inhibitors of the cAMP-dependent protein kinase, decreased basal ICa,L by about 85% and suppressed the effect of ACh. The ACh effect was also absent in cells dialysed with a non-hydrolysable analogue of cAMP, 8-bromo-cAMP. The results suggest that, in basal conditions, a large part of the L-type calcium channels should be phosphorylated by protein kinase A stimulated by a high cAMP level correlated with a high adenylate cyclase activity. The depressing effect of ACh on ICa,L may occur via inhibition of the high basal adenylate cyclase activity leading to a decrease of cAMP-dependent protein kinase stimulation and thus to a dephosphorylation of calcium channels.

Functional Expression of the TRPM4 Cationic Current in Ventricular Cardiomyocytes From Spontaneously Hypertensive Rats

Cardiac hypertrophy is associated with electrophysiological modifications, including modification of action potential shape that can give rise to arrhythmias. We report here a higher detection of a calcium-activated nonselective cation current in cardiomyocytes of spontaneously hypertensive rats (SHRs), a model of hypertension and heart hypertrophy when compared with Wistar-Kyoto (WKY) rat, its normotensive equivalent. Freshly isolated cells from the left ventricles of 3- to 6-month-old WKY rats or SHRs were used for patch-clamp recordings. In inside-out patches, the channel presented a linear conductance of 25±0.5 pS, did not discriminate Na+ over K+, and was not permeable to Ca2+. Open probability was increased by depolarization and a rise in [Ca2+]i (dissociation constant=10±5.4 &mgr;mol/L) but reduced by 0.5 mmol/L [ATP]i, 10 &mgr;mol/L glibenclamide, or flufenamic acid (IC50=5.5±1.7 &mgr;mol/L). Thus, it owns the fingerprint of the TRPM4 current. Although rarely detected in WKY cardiomyocytes, the current was present in >50% of patches from SHR cardiomyocytes. Moreover, by performing RT-PCR from ventricular samples, we observed that TRPM4 mRNA detection was higher in SHRs than in WKY rats. We propose that a TRPM4 current is expressed in ventricular cardiomyocytes from SHRs. According to its properties, this channel may contribute to the transient inward current implicated in delayed-after-depolarizations observed during [Ca2+] overload of cardiomyocytes.

Characterization of a Ca2+-activated Nonselective Cation Channel during Dedifferentiation of Cultured Rat Ventricular Cardiomyocytes

Cardiac hypertrophy is associated with electrical activity modifications, including sustained depolarization, that lead to a propensity for arrhythmias. The ionic currents underlying the sustained depolarization are not well defined. Similar modifications were reported on adult rat cardiomyocytes in primary culture undergoing dedifferentiation. Using the single-channel measurements on these cells, we identified the appearance of a Ca2+-activated nonselective cation channel (NSCCa) during the dedifferentiation process. In excised inside-out patches the channel presented a linear I/V relationship with a conductance of 26.5 pS. It was equally selective for Na+ and K+ and impermeable to Cl? and Ca2+ ions. The open probability increased with depolarization and with rise in intracellular calcium concentration. The channel activity was reduced by intracellular ATP and suppressed by flufenamic acid. Channel detection increased after incubation with a purinergic receptor agonist (ATPgS) or a PKC activator (PMA). Furthermore, occurrence of the channel developed during the culture. Absent at one day in vitro (d.i.v.), channel activity was present in 5, 46, 27 and 19% of patches after 4, 7, 14 and 21 d.i.v., respectively. We suggest that the channel may be associated with pro-arrhythmic signaling, in particular during the release of transmitters from autonomic nerve endings in the hypertrophied hearts.

A distinct de novo expression of Nav1.5 sodium channels in human atrial fibroblasts differentiated into myofibroblasts.

Fibroblasts play a major role in heart physiology. In pathological conditions, they can lead to cardiac fibrosis when they differentiate into myofibroblasts. This differentiated status is associated with changes in expression profile leading to neo‐expression of proteins such as ionic channels. The present study investigates electrophysiological changes associated with fibroblast differentiation focusing on voltage‐gated sodium channels in human atrial fibroblasts and myofibroblasts. We show that human atrial fibroblast differentiation in myofibroblasts is associated with de novo expression of voltage gated sodium current. Multiple arguments support that this current is predominantly supported by the Nav1.5 α‐subunit which may generate a persistent sodium entry into myofibroblasts. Our data revealed that Nav1.5 α‐subunit expression is not restricted to cardiac myocytes within the atrium. Since fibrosis is one of the fundamental mechanisms implicated in atrial fibrillation, it is of great interest to investigate how this channel could influence myofibroblasts function.

Dexrazoxane protects the heart from acute doxorubicin‐induced QT prolongation: a key role for IKs

Introduction:  Doxorubicin, an anthracycline widely used in the treatment of a broad range of tumours, causes acute QT prolongation. Dexrazoxane has been shown to prevent the QT prolongation induced by another anthracycline, epirubicin, but has not yet been reported to prevent that induced by doxorubicin. Thus, the present study was designed to test whether the acute QT effects induced by doxorubicin could be blocked by dexrazoxane and to explore the mechanism. Results were compared with those obtained with a reference human ether‐a‐go‐go (hERG) channel blocker, moxifloxacin.

Characterization of a hyperpolarization-activated current in dedifferentiated adult rat ventricular cells in primary culture

1 The presence of a hyperpolarization‐activated pacemaker (If)‐like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole‐cell patch clamp technique. 2 An If‐like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from ‐92.0 ± 2.5 and ‐63.0 ± 1.0 mV at the beginning (4‐day‐cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at ‐170 mV progressively increased from ‐2.34 ± 0.36 to ‐6.12 ± 0.64 pA pF−1 between the two farthest stages of cellular remodelling. In freshly isolated cells the current was activated at ‐108.0 ± 1.5 mV and its current density measured at ‐170 mV was ‐1.97 ± 0.56 pA pF−1. 3 The current was blocked by extracellular CsCl (3 mM) in a voltage‐dependent manner. Modification of reversal potentials obtained at various values of [K+]o (5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions. 4 It is concluded that the hyperpolarization‐activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of If. In adult rat ventricular cells it is activated in a non‐physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.

Activation of the Ca2+-Activated Nonselective Cation Channel by Diacylglycerol Analogues in Rat Cardiomyocytes

Introduction: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture.