Dario DiFrancesco

Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node.

Individual cells were isolated from the sino‐atrial node area of the rabbit heart using an enzyme medium containing collagenase and elastase. After enzymatic treatment the cells were placed in normal Tyrode solution, where beating resumed in a fraction of them. Isolated cells were studied in the whole cell configuration. Action potentials as well as membrane currents under voltage‐clamp conditions were similar to those in multicellular preparations. Pulses to voltages more negative than about ‐50 mV caused activation of the hyperpolarizing‐activated current, if. Investigation of the properties of this current was carried out under conditions that limited the influence of other current systems during voltage clamp. The if current activation range usually extended approximately from ‐50 to ‐100 mV, but varied from cell to cell. In several cases, pulsing to the region of ‐40 mV elicited a sizeable if. Both current activation and deactivation during voltage steps had S‐shaped time courses. A high variability was however observed in the sigmoidal behaviour of if kinetics. Plots of the fully‐activated current‐voltage (I‐V) relation in different extracellular Na and K concentrations showed that both ions carry the current if. While changes in the external Na concentration caused the current I‐V relation to undergo simple shifts along the voltage axis, changes in extracellular K concentration were also associated with changes in its slope. Again, a large variability was observed in the increase of I‐V slope on raising the external K concentration. The current if was strongly depressed by Cs, and the block induced by 5 mM‐Cs was markedly voltage dependent. Adrenaline (1‐5 microM) and noradrenaline (1 microM) increased the current if around the half‐activation voltage range and accelerated its activation at more negative voltages. Often, however, drug application failed to elicit any modification of if. Current run‐down was observed in nearly all cells, although at a highly variable rate. It was accelerated by raising the extracellular K concentration but did not show a marked use dependence. Both the if activation curve and the fully activated I‐V relation were affected by run‐down, the former being shifted to more negative values along the voltage axis and the latter being depressed with no apparent change of the if reversal potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Heart rate lowering by specific and selective If current inhibition with ivabradine: A new therapeutic perspective in cardiovascular disease

Resting heart rate is associated with cardiovascular and all-cause mortality, and the mortality benefit of some cardiovascular drugs seems to be related in part to their heart rate-lowering effects. Since it is difficult to separate the benefit of heart rate lowering from other actions with currently available drugs, a ‘pure’ heart rate-lowering drug would be of great interest in establishing the benefit of heart rate reduction per se.Heart rate is determined by spontaneous electrical pacemaker activity in the sinoatrial node. Cardiac pacemaker cells generate the spontaneous slow diastolic depolarisation that drives the membrane voltage away from a hyperpolarised level towards the threshold level for initiating a subsequent action potential, generating rhythmic action potentials that propagate through the heart and trigger myocardial contraction. The If current is an ionic current that determines the slope of the diastolic depolarisation, which in turn controls the heart beating rate.Ivabradine is the first specific heart rate-lowering agent to have completed clinical development for stable angina pectoris. Ivabradine specifically blocks cardiac pacemaker cell f-channels by entering and binding to a site in the channel pore from the intracellular side. Ivabradine is selective for the If current and exerts significant inhibition of this current and heart rate reduction at concentrations that do not affect other cardiac ionic currents. This activity translates into specific heart rate reduction, which reduces myocardial oxygen demand and simultaneously improves oxygen supply, by prolonging diastole and thus allowing increased coronary flow and myocardial perfusion. Ivabradine lowers heart rate without any negative inotropic or lusitropic effect, thus preserving ventricular contractility.Ivabradine was shown to reduce resting heart rate without modifying any major electrophysiological parameters not related to heart rate. In patients with left ventricular dysfunction, ivabradine reduced resting heart rate without altering myocardial contractility. Thus, pure heart rate lowering can be achieved in the clinic as a result of specific and selective If current inhibition.Two randomised clinical studies have shown that ivabradine is an effective anti-ischaemic agent that reduces heart rate and improves exercise capacity in patients with stable angina. Ivabradine was shown to be superior to placebo in improving exercise tolerance test (ETT) criteria (n = 360) and, in a 4-month, double-blind, controlled study (n = 939), ivabradine 5 and 7.5mg twice daily were shown to be at least as effective as atenolol 50 and 100mg once daily, respectively, in improving total exercise duration and other ETT criteria, and reducing the number of angina attacks.Experimental data indicate a potential role of pure heart rate lowering in other cardiovascular conditions, such as heart failure.

The Role of the Funny Current in Pacemaker Activity

Pacemaking is a basic physiological process, and the cellular mechanisms involved in this function have always attracted the keen attention of investigators. The “funny” (If) current, originally described in sinoatrial node myocytes as an inward current activated on hyperpolarization to the diastolic range of voltages, has properties suitable for generating repetitive activity and for modulating spontaneous rate. The degree of activation of the funny current determines, at the end of an action potential, the steepness of phase 4 depolarization; hence, the frequency of action potential firing. Because If is controlled by intracellular cAMP and is thus activated and inhibited by &bgr;-adrenergic and muscarinic M2 receptor stimulation, respectively, it represents a basic physiological mechanism mediating autonomic regulation of heart rate. Given the complexity of the cellular processes involved in rhythmic activity, an exact quantification of the extent to which If and other mechanisms contribute to pacemaking is still a debated issue; nonetheless, a wealth of information collected since the current was first described more than 30 years ago clearly agrees to identify If as a major player in both generation of spontaneous activity and rate control. If- dependent pacemaking has recently advanced from a basic, physiologically relevant concept, as originally described, to a practical concept that has several potentially useful clinical applications and can be valuable in therapeutically relevant conditions. Typically, given their exclusive role in pacemaking, f-channels are ideal targets of drugs aiming to pharmacological control of cardiac rate. Molecules able to bind specifically to and block f-channels can thus be used as pharmacological tools for heart rate reduction with little or no adverse cardiovascular side effects. Indeed a selective f-channel inhibitor, ivabradine, is today commercially available as a tool in the treatment of stable chronic angina. Also, several loss-of-function mutations of HCN4 (hyperpolarization-activated, cyclic-nucleotide gated 4), the major constitutive subunit of f-channels in pacemaker cells, are known today to cause rhythm disturbances, such as for example inherited sinus bradycardia. Finally, gene- or cell-based methods for in situ delivery of f-channels to silent or defective cardiac muscle represent novel approaches for the development of biological pacemakers eventually able to replace electronic devices.

A new interpretation of the pace‐maker current in calf Purkinje fibres.

1. The properties of the ‘pace‐maker’ current iK2 of Purkinje fibres are investigated to verify whether its behaviour during voltage‐clamp pulses is consistent with the view that iK2 is a K current deactivating on hyperpolarization in the range ‐50 to ‐100 mV. 2. During voltage‐clamp pulses in the range positive to the apparent reversal potential (Erev), low concentrations of Cs depress the time‐dependent current change without altering the time‐independent current component. The total current becomes more outward in Cs, which on the assumption that Cs only affects the iK1 and iK2 channels implies that iK2 is inward in the voltage range analysed. 3. Ba strongly reduces the time‐independent component due to iK1 and therefore limits the contribution of K depletion to the total current time course during hyperpolarizations. In the presence of barium a current reversal is not obtained even with large hyperpolarizations in normal Tyrode solution. If in Ba‐containing solutions the external K concentration is raised from 3 up to 48 mM, iK2 greatly increases in the inward direction during hyperpolarizations in the range ‐51 to ‐101 mV, implying that it cannot be carried by K only. 4. In the presence of Ba, measurements of the membrane conductance due to iK2 indicate a channel‐opening process during hyperpolarizations and a channel‐closing process during depolarizations, in the K‐concentration range analysed. 5. It is concluded that iK2 is not, as had been previously thought, a pure K current, but is rather an inward current activated during hyperpolarizations negative to about ‐50 mV. This provides further evidence for a possible identity between iK2 in Purkinje fibres and the current if in the SA node.

A study of the ionic nature of the pace-maker current in calf Purkinje fibres.

1. Properties of the pace‐maker current (if) in Purkinje fibres were studied in the presence of Ba, which by partially blocking the iK1 channel reduces K depletion during hyperpolarizing voltage‐clamp pulses, and eliminates the main cause of distortion in the current time course. 2. On raising the external potassium concentration (Kb), the if fully activated current‐‐voltage relation (if(E)) increases in the inward direction. In the range 3‐‐36 mM‐‐Kb and negative to ‐50 mV the current is inward, and no cross‐over is observed. 3. In normal conditions, the reversal potential (Ef) for if lies in the voltage region positive to ‐50 mV, and can be observed on lowering the external sodium concentration (Nab). Ef shifts to the negative direction when Nab is decreased. Slopes ranging between 29 and 35 mV/decade are found for Nernst plots of Ef against Nab. Changing Nab in the range 140‐‐4.4 mM causes the if(E) relation to undergo a simple shift along the voltage axis, without significant change in its slope. 4. Ef also depends on Kb, as can be observed in low Nab (35 mM), and shifts to the positive direction by about 26 mV for every 10‐fold change in Kb. The fully activated slope conductance increases when Kb is increased. 5. It is concluded that Na and K both participate in carrying if. The slope of the fully activated if(E) relation increases with Kb, but is unchanged in different Nab, indicating that the channel conductance depends on Kb, but not appreciably on Nab.

Voltage‐clamp investigations of membrane currents underlying pace‐maker activity in rabbit sino‐atrial node.

1. Small preparations of spontaneously beating rabbit sino‐atrial node have been investigated using the two micro‐electrode voltage‐clamp technique. 2. Hyperpolarizing clamp pulses given from holding potentials of about ‐45 mV reveal a time‐dependent change of a membrane current, if, which is shown to overlap the pace‐maker range (‐65 mV to ‐45 mV) for these preparations. 3. The changes in if are shown to be quite distinct from the de‐activation of the time‐dependent outward current, iK. 4. The time‐dependent changes of the if system increase during adrenaline application and therefore contribute to the chronotropic action of adrenaline on the heart. 5. Evidence for a link between slow inward current (iSi) and time‐dependent outward current (iK) in rabbit sino‐atrial node is presented and assessed.

Molecular Architecture of the Human Sinus Node Insights Into the Function of the Cardiac Pacemaker

Background— Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. Methods and Results— Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Nav1.5, Kv4.3, Kv1.5, ERG, Kir2.1, Kir6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Cav1.3, Cav3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of Kv4.2, Kir6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. Conclusions— Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Current-dependent Block of Rabbit Sino-Atrial Node I f Channels by Ivabradine

“Funny” (f-) channels have a key role in generation of spontaneous activity of pacemaker cells and mediate autonomic control of cardiac rate; f-channels and the related neuronal h-channels are composed of hyperpolarization-activated, cyclic nucleotide–gated (HCN) channel subunits. We have investigated the block of f-channels of rabbit cardiac sino-atrial node cells by ivabradine, a novel heart rate-reducing agent. Ivabradine is an open-channel blocker; however, block is exerted preferentially when channels deactivate on depolarization, and is relieved by long hyperpolarizing steps. These features give rise to use-dependent behavior. In this, the action of ivabradine on f-channels is similar to that reported of other rate-reducing agents such as UL-FS49 and ZD7288. However, other features of ivabradine-induced block are peculiar and do not comply with the hypothesis that the voltage-dependence of block is entirely attributable to either the sensitivity of ivabradine-charged molecules to the electrical field in the channel pore, or to differential affinity to different channel states, as has been proposed for UL-FS49 (DiFrancesco, D. 1994. Pflugers Arch. 427:64–70) and ZD7288 (Shin, S.K., B.S. Rotheberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91–101), respectively. Experiments where current flows through channels is modified without changing membrane voltage reveal that the ivabradine block depends on the current driving force, rather than voltage alone, a feature typical of block induced in inwardly rectifying K+ channels by intracellular cations. Bound drug molecules do not detach from the binding site in the absence of inward current through channels, even if channels are open and the drug is therefore not “trapped” by closed gates. Our data suggest that permeation through f-channel pores occurs according to a multiion, single-file mechanism, and that block/unblock by ivabradine is coupled to ionic flow. The use-dependence resulting from specific features of If block by ivabradine amplifies its rate-reducing ability at high spontaneous rates and may be useful to clinical applications.

The contribution of the ‘pacemaker’ current (if) to generation of spontaneous activity in rabbit sino-atrial node myocytes.

1. The contribution to the diastolic depolarization of the hyperpolarization‐activated current, if, relative to other components was investigated in isolated rabbit sino‐atrial (SA) node myocytes. 2. During the diastolic phase the membrane potential depolarized by 0.1096 +/‐ 0.014 V/s, which requires only about 3 pA of inward current in a cell with an average capacity of 30 pF. The problem of which ionic component is responsible for initiating the diastolic depolarization was investigated by analysing the composition and the properties of the net inward current in the diastolic range of voltages. 3. The measured instantaneous ‘background’ current activated during voltage clamp steps from a holding potential of ‐35 mV was outward positive to approximately ‐61 mV, and had a region of negative slope conductance from ‐45 to ‐35 mV. 4. The instantaneous component lost its rectifying behaviour in the presence of Ni2+ (100 microM) and nitrendipine (10 microM). These blockers of Ca(2+)‐dependent currents modified the instantaneous I‐V relation at voltages positive to ‐45 to ‐50 mV, thus implying that Ca2+ currents become important at less negative potentials than ‐50 mV, towards the very end of diastolic depolarization. 5. Possible errors introduced by voltage clamp analysis with the whole‐cell method on the instantaneous current and on if measurement were evaluated. Leakage through the seal resistance caused the instantaneous I‐V relation to be displaced in the inward direction at negative voltages. Correction for the seal leakage moved the reversal potential for the instantaneous current toward the negative direction from ‐61 to approximately ‐66 mV. Thus, no depolarization can be driven by the background current beyond ‐66 mV. 6. During voltage clamp analysis, lack of series‐resistance compensation led to lack of intracellular voltage control, as was apparent using a second pipette on the same cell. This slowed activation of if and led to a 1.5‐ to 2‐fold reduction of if size in the range ‐55 to ‐115 mV. Thus, uncorrected measurements of the instantaneous component and of if may concur to underestimate the role of if in pacemaking. 7. These results lead to the conclusion that in the SA node cells analysed, pacemaker activity is generated with the essential contribution of the hyperpolarization‐activated current, if. Numerical computation of SA node cell activity using an extension of the DiFrancesco‐Noble model shows that the if‐activation hypothesis can account for the presence of spontaneous action potentials and their sensitivity to if changes.

What keeps us ticking: a funny current, a calcium clock, or both?

The processes underlying the initiation of the heartbeat, whether due to intracellular metabolism or surface membrane events, have always been a major focus of cardiac research. About 50 years ago, a pioneering work initiated by Silvio Weidmann and others applied the Hodgkin–Huxley formalism of membrane excitation to interpret the cardiac electrical activity, including the pacemaker depolarization (see D. Noble, 1979. The initiation of the heartbeat, Clarendon Press). The underlying idea was that voltageand time-dependent gating of various surface membrane channels not only generated the cardiac pacemaker action potential (AP), but also controlled the spontaneous depolarization between APs and thus determinedwhen thenextAPwouldoccur. According to this description, the ensemble of surface membrane ion channels works as a clock that regulates the rate and rhythm of spontaneous AP firing, otherwise known as normal automaticity. A formidable research effort then concentrated in attempting to target which of the surface membrane ion channels had an important role in controlling the spontaneous diastolic depolarization (DD). Originally, a major role was attributed to the “IK-decay theory”. This was strongly influenced by the previous Hodgkin–Huxley model of nerve AP, which described the slow depolarization following a nerve AP as due to the decay of a K current. This model of pacemaker depolarization lasted some 20 years, until it was turned upside-down by a full re-interpretation based on the discovery of the If current. Other ionic currents gated by membrane depolarization, i.e. ICaL, ICaT, IST, non-gated and non-specific background leak currents, and also a current generated by the Na–Ca exchange (NCX) carrier, were also proposed to be involved in pacemaking. Based on a wealth of experimental evidence, If is today considered as the most important ion channel involved in the rate regulation of cardiac pacemaker cells, and is sometimes referred to as “the pacemaker channel.” Several studies, some of which recent, have also shown that in addition to voltage and time, surface membrane electrogenic molecules are strongly modulated by Ca and phosphorylation. The studies of a sub-group of pacemaker cell researchers focusing Journal of Molecular and Cellular Cardiology 47 (2009) 157–170