Ionic channels underlying cardiac automaticity: new insights from genetically-modified mouse strains.

The spontaneous activity (or pacemaker activity) of the heart constitutes a fundamental physiological function in higher organisms. Pacemaker activity is generated in the sino-atrial node (SAN) by a specialized cell population adapted to the generation of a rhythmic electrical oscillation. The precise ionic mechanisms underlying initiation of pacemaking in automatic cells has not been entirely elucidated. Ionic channels and intracellular Ca2+ signalling in pacemaker cells are both required for the proper setting of pacemaking. Understanding the mechanisms of pacemaker activity is important for developing new therapeutic approaches for controlling the heart rate in the diseased myocardium. Controlling the heart rate in the clinical practice is a promising way to increase cardioprotection and improve patient's survival in cardiac ischemic pathology. We describe here the contribution of several ion channels families into the generation and regulation of the heart rate using new approaches involving genetically modified mouse strains. These studies underline the functional redundancy of mechanisms underlying pacemaking, an important safety parameter for new drugs targeting ion channels to modulate cardiac frequency.

Physiological and pharmacological insights into the role of ionic channels in cardiac pacemaker activity.

The generation of cardiac pacemaker activity is a complex phenomenon which requires the coordinated activity of different membrane ionic channels, as well as intracellular signalling factors including Ca(2+) and second messengers. The precise mechanism initiating automaticity in primary pacemaker cells is still matter of debate and certain aspects of how channels cooperate in the regulation of pacemaking by the autonomic nervous system have not been entirely elucidated. Research in the physiopathology of cardiac automaticity has also gained a considerable interest in the domain of cardiovascular pharmacology, since accumulating clinical and epidemiological evidence indicate a link between an increase in heart rate and the risk of cardiac mortality and morbidity. Lowering the heart rate by specific bradycardic agents in patients with heart disease constitutes a promising way to increase cardioprotection and improve survival. Thus, the elucidation of the mechanisms underlying the generation of pacemaker activity is necessary for the development of new therapeutic molecules for controlling the heart rate. Recent work on genetically modified mouse models provided new and intriguing evidence linking the activity of ionic channels genes to the generation and regulation of pacemaking. Importantly, results obtained on genetically engineered mouse strains have demonstrated that some channels are specifically involved in the generation of cardiac automaticity and conduction, but have no functional impact on the contractile activity of the heart. In this article, we will outline the current knowledge on the role of ionic channels in cardiac pacemaker activity and suggest new potential pharmacological targets for controlling the heart rate without concomitant negative inotropism.

If current inhibition: cellular basis and physiology.

The slow diastolic depolarization phase in cardiac pacemaker cells is the electrical basis of cardiac automaticity. The hyperpolarization-activated current (I(f)) is one of the key mechanisms underlying diastolic depolarization. Particularly, I(f) is unique in being activated on membrane hyperpolarization following the repolarization phase of the action potential. I(f) has adapted biophysical properties and voltage-dependent gating to initiate pacemaker activity. I(f) possibly constitutes the first voltage-dependent trigger of the diastolic depolarization. For these reasons, I(f) is a natural pharmacological target for controlling heart rate in cardiovascular disease. In this view, I(f) inhibitors have been developed in the past, yet the only molecule to have reached the clinical development is ivabradine. At the cellular level, the remarkable success of ivabradine is to be ascribed to its relatively high affinity for f-channels. Furthermore, ivabradine is the most I(f)-specific inhibitor known to date, since moderate inhibition of other voltage-dependent ionic currents involved in automaticity can be observed only at very high concentrations of ivabradine, more than one order of magnitude from that inhibiting I(f). Finally, the mechanism of block of f-channels by ivabradine has particularly favorable properties in light of controlling heart rate under variable physiological conditions. In this article, we will discuss how I(f) inhibition by ivabradine can lead to reduction of heart rate. To this aim, we will comment on the role of I(f) in cardiac automaticity and on the mechanism of action of ivabradine on f-channels. Some aspects of the cardiac pacemaker mechanism that improve the degree of security of ivabradine will also be highlighted.

Properties of the hyperpolarization-activated current (I(f)) in isolated mouse sino-atrial cells.

OBJECTIVE: We have investigated the properties of the hyperpolarization-activated (I(f)) current in pacemaker cells from the mouse sino-atrial node (SAN). METHODS: The I(f) current was studied in cells isolated enzymatically from the SAN region of adult C57BL6/J mice. The whole-cell variation of the patch-clamp technique was employed to investigate the basic properties of I(f). RESULTS: In mouse SAN cells, the I(f) current density at -120 mV was 18+/-2 pA/pF (n=23). I(f) was not detected in cells showing atrial-like morphology that were also found in SAN preparations (n=7). I(f) was blocked by 5 mM Cs(+), was inhibited by application of 5 microM acetylcholine, and was increased by 10 microM noradrenaline. The I(f) current reversal potential was -31+/-2 mV under physiological concentration of Na(+) and K(+) ions. Lowering the extracellular Na(+) concentration reduced I(f) amplitude, while increased when the extracellular K(+) concentration was augmented. I(f) voltage for half activation was -87+/-1 mV (n=6). CONCLUSIONS: We conclude that the native I(f) current in mouse SAN cells shows functional properties that are similar to I(f) described in rabbit SAN tissue. This study opens the possibility of investigating the involvement of I(f) in the regulation of heart rate in genetically modified mice.

Inhibition of T-type and L-type calcium channels by mibefradil: physiologic and pharmacologic bases of cardiovascular effects.

Ca2+ channel antagonists of the dihydropyridine, benzothiazepine, and phenylalkylamine classes have selective effects on L-type versus T-type Ca2+ channels. In contrast, mibefradil was reported to be more selective for T-type channels. We used the whole-cell patch-clamp technique to investigate the effects of mibefradil on T-type and L-type Ca2+ currents (I(CaT) and I(CaL)) recorded at physiologic extracellular Ca2+ in different cardiac cell types. At a stimulation rate of 0.1 Hz, mibefradil blocked I(CaT) evoked from negative holding potentials (HPs) (-100 mV to -80 mV) with an IC50 of 0.1 microM in rat atrial cells. This concentration had no effect on I(CaL) in rat ventricular cells (IC50: approximately3 microM). However, block of I(CaL) was enhanced when the HP was depolarized to -50 mV (IC50: approximately 0.1 microM). Besides a resting block, mibefradil displayed voltage- and use-dependent effects on both I(CaT) and I(CaL). In addition, inhibition was enhanced by increasing the duration of the step-depolarizations. Similar effects were observed in human atrial and rabbit sinoatrial cells. In conclusion, mibefradil combines the voltage- and use-dependent effects of dihydropyridines and benzothiazepines on I(CaL). Inhibition of I(CaL), which has probably been underestimated before, may contribute to most of the cardiovascular effects of mibefradil.

Cyclosporin A increases basal intracellular calcium and calcium responses to endothelin and vasopressin in human coronary myocytes.

Cyclosporin A (CsA) is a widely used immunosuppressive agent with severe side effects including hypertension. Here, we investigated the effects of CsA on intracellular free calcium ([Ca(2+)](i)) and the mechanisms involved in vasoconstriction in cultured human coronary myocytes. We used the Fura-2 technique for Ca(2+) imaging. Acute application of CsA at therapeutic concentrations (0.1-10 micromol/l) had no effect. Chronic exposure to CsA (1 micromol/l) for 24 h induced a small (20 nmol/l) but highly significant increase of basal [Ca(2+)](i) and enhanced the occurrence of spontaneous Ca(2+) oscillations. Endothelin- and vasopressin-induced rises of [Ca(2+)](i) were also enhanced. The demonstration that CsA increases basal [Ca(2+)](i) in addition to its impact on agonist receptor stimulation is of major importance for new therapeutic approaches.

Facilitation of the L-type calcium current in rabbit sino-atrial cells: effect on cardiac automaticity.

OBJECTIVE: The L-type Ca(2+) current (I(Ca,L)) contributes to the generation and modulation of the pacemaker action potential (AP). We investigated facilitation of I(Ca,L) in sino-atrial cells. METHODS: Facilitation was studied in regularly-beating cells isolated enzymatically from young albino rabbits (0.8-1 kg). We used the whole-cell patch-clamp technique to vary the frequency of the test depolarizations evoked at -10 mV or the conditioning diastolic membrane potential prior to the test pulse. RESULTS: High frequencies (range 0.2-3.5 Hz) slowed the decay kinetics of I(Ca,L) evoked from a holding potential (HP) of -80 mV in 68% of cells resulting in a larger Ca(2+) influx during the test pulse. The amount of facilitation increased progressively between 0.2 and 3.0 Hz. When the frequency was changed from 0.1 to 1 Hz, the averaged increase in the time integral of I(Ca,L) was 27+/-7% (n=22). Application of conditioning voltages between -80 and -50 mV induced similar facilitation of I(Ca,L) in 73% of cells. The maximal increase of Ca(2+) entry occurred between -60 and -50 mV, and was on average 38+/-14% for conditioning prepulses of 5 s in duration (n=15). Numerical simulations of the pacemaker activity showed that facilitation of I(Ca,L) promotes stability of sino-atrial rate by enhancing Ca(2+) entry, thus establishing a negative feedback control against excessive heart rate slowing. CONCLUSION: Facilitation of I(Ca,L) is present in rabbit sino-atrial cells. The underlying mechanism reflects modulation of I(Ca,L) decay kinetics by diastolic membrane potential and frequency of depolarization. This phenomenon may provide an important regulatory mechanism of sino-atrial automaticity.

Evidence for tetrodotoxin-sensitive sodium currents in primary cultured myocytes from human, pig and rabbit arteries.

Primary cultured human coronary myocytes express a tetrodotoxin-sensitive sodium current (I(Na)). Here, we have investigated whether I(Na) is expressed in vascular smooth muscles cells (VSMCs) isolated from other large arteries, and other mammals. VSMCs were enzymatically dissociated, kept in primary culture, and macroscopic I(Na) was recorded using the whole-cell patch-clamp technique. We found that I(Na) is expressed in VSMCs grown from human aortic (90%; n=50) and pulmonary (44%; n=19) arteries, and in the human aortic myocyte cell line HAVSMC (94%; n=27). I(Na) was also detected in pig coronary (60%; n=33), and rabbit aortic (47%; n=15), but not in rat aortic VSMCs (n=20). These different I(Na) had similar voltage thresholds for activation (approximately equal to -50 mV), and were highly sensitive to extracellularly applied tetrodotoxin. We conclude that I(Na) is expressed in VSMCs grown from various types of large arteries in humans, pig and rabbit.

Characterisation of alpha 1A Ba2+, Sr2+ and Ca2+ currents recorded with the ancillary beta 1-4 subunits.

Xenopus oocytes have been injected with different combinations of expression plasmids carrying the rat brain alpha 1A and different beta (beta 1-4) Ca2+ channel subunit cDNAs. Whole-cell Ba2+ and Ca2+ currents were recorded up to seven days after injection. Intra-oocyte injection of BAPTA allowed us to record uncontaminated Ba2+, Sr2+ currents. The alpha 1A calcium channel showed relative current amplitudes according to the sequence: IBa2+ > ISr2+ > ICa2+. The ratio ICa2+/IBa2+ was significantly larger when compared to the class C L-type Ca2+ channel (alpha 1C). However, currents flowing through alpha 1A and alpha (1C) subunits saturate for similar Ba2+ concentrations and display the anomalous mole fraction effect in the presence of mixtures of Ba2+ and Ca2+ ions in the external medium. In oocytes expressing the alpha 1A Ca2+ channel subunit, switching from extracellular Ba2+ to Ca2+ also induced a depolarising shift of current-to-voltage relation and the steady-state inactivation curve, and increased the time-to-peak of the current. Inactivation kinetics were poorly affected. Changes in gating and voltage-dependence of activation, but not in the voltage-dependent inactivation, were independent from the coexpressed beta subunit (except with the beta 4 subunit). Our data constitute strong evidence for the existence of differences in intra-pore Ca2+ binding sites between the alpha 1C and alpha 1A subunits, and emphasise the influence of the charge carrier on the modulation of alpha 1A properties by the beta subunits.

Modulation of the alpha 1A Ca2+ channel by beta subunits at physiological Ca2+ concentration.

The class A Ca2+ channel alpha 1 subunit (alpha 1A) was expressed in Xenopus oocytes alone or in combination with the beta 1b, beta 2a, beta 3, or beta 4 subunit. Analysis of voltage-dependent activation and inactivation in the presence of 1.8 mM external Ca2+ showed an hyperpolarising shift of both relations when compared to similar recordings performed in the presence of 40 mM Ba2+. These shifts, which differed for activation and inactivation, were strongly modulated by the nature of the coexpressed beta subunit. On the other hand, for each combination, the kinetics of inactivation were similar in 1.8 mM Ca2+ and 40 mM Ba2+ (for example co-expression of the beta 2a subunit reduced inactivation using either 40 mM Ba2+ or 1.8 mM Ca2+). Thus, modulation of channel properties by the beta subunit is different in physiological Ca2+ or high Ba2+ concentrations. These results must be taken into consideration to extrapolate the role of the beta subunit in native cells.

Coexpression of the beta2 subunit does not induce voltage-dependent facilitation of the class C L-type Ca channel.

Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of the alpha1 and beta subunits of the alpha1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation in Xenopus oocytes. Here we show that, by expressing the alpha1C together with different beta subunits in oocytes, the beta1, beta3 and beta4, but not the beta2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of the beta2 subunit mRNA, suggest that beta2 may be the beta subunit associated with functional cardiac L-type Ca2+ channels.

Change in membrane permeability induced by protegrin 1: implication of disulphide bridges for pore formation.

Protegrin 1 (PG-1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG-1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.