Download I inhibition: a novel mechanism of action

European Heart Journal Supplements (2003) 5 (Supplement G), G19—G25
If inhibition: a novel mechanism of action
D. DiFrancesco
Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and
Neurobiology, Milan, Italy
KEYWORDS
Cardiac rate;
Funny current;
If blockers;
Pacemaker;
Sinoatrial node
Aims It is well established that the cardiac pacemaker (‘funny’, or If) current plays
an important role in the generation and autonomic modulation of cardiac rate by
controlling the rate of diastolic depolarization. Here, the properties of If and the
criteria that permit identification of If activation as the main mechanism responsible
for diastolic depolarization are briefly summarized. The relationship between If
inhibition by specific If channel blockers (rate-reducing agents) and reduction in
pacemaker rate is also described.
Methods and results The If data reported here were collected from rabbit sinoatrial
node cells that were isolated and patch-clamped. Cs+ ions and, more efficiently, ‘ratereducing’ agents block If and reduce the steepness of diastolic depolarization and
frequency in spontaneously active sinoatrial node myocytes. Ivabradine (Procoralan®;
Servier, Neuilly-sur-Seine, France), a recently developed molecule, blocks If channels
when they are open and preferentially when the current is outward.
Conclusions If controls the slope of diastolic depolarization and cardiac frequency,
and its inhibition causes heart rate reduction. The current-dependent blockade of If
with ivabradine leads to a specific and use-dependent, heart rate reducing effect
that may have therapeutic applications in clinical settings.
© 2003 The European Society of Cardiology. Published by Elsevier Science Ltd. All
rights reserved
Introduction
The ability of sinoatrial node pacemaker cells to
pace spontaneously depends on the presence in
these cells of the diastolic depolarization phase of
the action potential (phase 4). At the end of an
action potential, the repolarization process
terminates at a maximum diastolic depolarization
(MDP) level of around —60 to —70 mV; this level of
depolarization is greater than those that are typical
of working cardiac muscle (i.e. under —80 mV). The
membrane voltage does not set to rest, but slowly
Correspondence: Dario DiFrancesco, Department of General
Physiology and Biochemistry, Laboratory of Molecular
Physiology and Neurobiology, via Celoria 26, 20133 Milano,
Italy.
depolarizes until it arrives at the threshold for
activation of the faster transient, which leads to
another action potential, thus producing rhythmic
activity (Fig. 1). In the mammalian heart, diastolic
depolarization is typically found in cardiac regions
that are able to pace spontaneously and is absent
in the working myocardium, except under specific
pathological conditions. The diastolic depolarization, or pacemaker, phase of the action potential
serves two main purposes: first, it is responsible
for generation of spontaneous activity of the
sinoatrial node, and hence of cardiac rhythm; and
second, it mediates regulation of pacemaker
frequency by the autonomic nervous system.
Therefore, acquiring a better understanding of the
ionic and cellular mechanisms that underlie
generation of pacemaker depolarization, and more
01520-765X/03/0G0019 + 07 $35.00/0 © 2003 The European Society of Cardiology, Published by Elsevier Science Ltd. All rights reserved.
G20
D. DiFrancesco
(a) Iso 0.3 µM
control
control
ACh 0.03 µM
100 ms
100 ms
50
mV
50
mV
(b)
—35 100 ms
—35 100 ms
control
500
pA
250
pA
—85
Iso 1 µM
(c)
Iso 1 µM
control
mV —100 —50
ACh 0.3 µM
—85
control
1.0
0.5
0
control
ACh 1 µM
mV —100 —50
1.0
0.5
0
Fig. 1
If mediates chronotropic modulation of pacemaker
activity by the autonomic transmitters. (a) Spontaneous action
potentials recorded in two isolated sinoatrial node myocytes in
control conditions and during perfusion with isoprenaline (Iso)
0.03 µmol/l (left) or acethylcholine (ACh) 0.03 µmol/l (right),
as indicated. Note acceleration by Iso and slowing by ACh. (b)
If records obtained during hyperpolarization to —85 mV from a
holding potential of —35 mV in two different cells in control
Tyrode solution and during perfusion with Iso 1 µmol/l (left) or
ACh 0.3 µmol/l (right). Note that If increased with Iso and
decreased with ACh. (c) The If activation curve shifted by about
13.0 mV in the presence of Iso 1 µmol/l (left), and in a different cell by about —10.6 mV in the presence of ACh 1 µmol/l
(right). Activation curves were measured by a voltage ramp
method on the left13 and by a voltage step activation protocol
on the right.2 All records were taken at a temperature of 35°C.
generally of pacemaker activity, remains a central
issue in cardiac physiology. Because rhythmic
firing is a basic type of activity of other cell types,
typically of neurones, this issue is in fact central
to physiology as a whole.
What is known of the mechanisms that underlie
pacemaker generation? The MDP is depolarized in
sinoatrial node cells; furthermore, at the basis of
repetitive activity is a slowly progressing
depolarization. Those observations indicate that
an inward current component must be expressed
specifically in these cells and be activated during
the slow depolarizing phase. An inward current
that is activated on hyperpolarization in the
diastolic range of voltages was indeed described
during the late 1970s, and was shown to have
properties sufficient for generating phase 4 of the
pacemaker cell action potential.1 The ‘funny’ (If)
current is so termed because of its unusual
features, including that of being an inward current
that is activated on hyperpolarization and not on
depolarization like other known currents. If was
found to be able not only to generate a slow
depolarization but also to control its slope, and
hence the rate of cardiac rhythm, under the
influence of adrenaline (epinephrine); in other
words, If was shown to mediate the positive
chronotropic action of beta-receptor stimulation
by sympathetic stimuli.2
Early studies of pacemaker activity in the
conduction tissue (Purkinje fibres) had already led
to the identification of a ‘pacemaker’ current.
However, this had been erroneously interpreted as
a pure K+ current (IK2), outward and deactivating
on hyperpolarization in the pacemaker range of
voltages.3 This inaccurate description held until
the early 1980s, when the discovery of If in the
sinoatrial node raised serious reservations as to
the possibility that two pacemaker currents of
different ionic nature could exist. This issue was
resolved in 1981 with the discovery that the
Purkinje fibre’s IK2 had been wrongly interpreted
and was indeed identical to the nodal If.4,5 Those
findings revealed the existence of a general
mechanism for inducing spontaneous activity in
cardiac cells.
Since its discovery, the If current has been the
object of several studies aimed at understanding its
ionic, kinetic and modulatory properties.6,7 If-like
currents were also described in neurones and shown
to be important to several aspects of the regulation
of neuronal excitability.8 Identification of the
molecular subunits contributing to form the native
pacemaker channels subsequently provided new
tools with which to investigate their molecular
properties. The newly cloned family of
hyperpolarization-activated, cyclic-nucleotidegated (HCN) channels comprises four isoforms that
are variously distributed among cardiac cells and
neurones.9—11 Here, the properties of native cardiac
pacemaker channels are briefly summarized, and
some basic aspects of channel blockade by specific
If inhibitors (rate-reducing agents) are discussed.
Methods
The experiments reported here were conducted in
sinoatrial node cells isolated from rabbit hearts
and
patch-clamped
in
the
whole-cell
configuration. The methods for isolation and
storage of single cells from the central sinoatrial
If inhibition: a novel mechanism of action
node area were reported in detail previously.12
The cells studied were perfused with normal
Tyrode solution under the stage of an inverted
microscope and were patch-clamped with pipettes
filled with an intracellular-like solution. Control
and test solutions were delivered via a fastperfusion pipette located on top of the cell under
study. The compositions of the perfusing and
pipette solutions are described elsewhere.13
Temperature was controlled in all experiments and
was set to between 32 and 35°C. Data analysis was
performed off-line.
Results and discussion
The If current and pacemaker activity
The questions as to whether If is indeed
responsible
for
generation
of
diastolic
depolarization and the extent to which it
participates in the control of cardiac rate have
been subject to intense debate since the current
was first described in the sinoatrial node.7,14
An indication of the possible functions of If can
easily be derived from its elementary
properties.7,12 If is a mixed Na+ and K+ current that
is activated on hyperpolarization to voltages
within the pacemaker range. Its reversal potential
in normal Tyrode solution is approximately —10 to
—20 mV, and the current is therefore inward at
diastolic voltages. Its activation threshold is about
—40 to —50 mV, and the current is fully activated
at about —100 to —110 mV. If activates slowly upon
hyperpolarization, with a time constant that
becomes shorter at more negative voltages (about
1 s at —55 mV and 0.5 s at —75 mV), and it
deactivates rapidly upon depolarization to
voltages within the plateau range (about 30 ms at
15 mV).15 Based on these features, it is possible to
understand how If works. Because the activation
range of If overlaps that of diastolic depolarization
(Fig. 1a and c), repolarization of the action
potential will lead to If activation; and because If is
inward at diastolic voltages, its activation will lead
to generation of a slowly developing depolarization
process — the diastolic depolarization.
Together with its elementary properties, several
other experimental findings reported since the
discovery of If contribute to a wealth of evidence
highlighting the fundamental role of this current in
the spontaneous activity of cardiac pacemaker
cells, as well as in the control of various aspects of
cell excitability, including spontaneous firing of
neurones.7,8 Among those findings, especially
relevant is the modulation of cardiac rate by
G21
autonomic transmitters, in which the basic role of
If has been demonstrated, and the evidence that
pharmacological blockade of If channels with
specific blocking molecules affects the rate of
diastolic depolarization, and hence cardiac rate,
in a rather specific manner. These aspects are
discussed below.
Heart rate is controlled by If
The sinoatrial node, or pacemaker, region of the
mammalian heart is the most densely innervated
by both adrenergic and cholinergic branches of the
autonomic nervous system. Through innervation of
the sinoatrial node, the autonomic nervous system
exerts control over the chronotropic state of the
heart; sympathetic stimulation accelerates and
parasympathetic stimulation slows cardiac
frequency by acting through beta-adrenergic and
muscarinic receptors, respectively. What are the
cellular mechanisms that are responsible for the
autonomic modulation of rate? Low sympathetic
and parasympathetic agonist concentrations
modify the rate via changes in the steepness of
diastolic depolarization.
In Fig. 1a, the effects of beta-adrenergic (left)
and muscarinic stimulation (right) on spontaneous
activity recorded from isolated sinoatrial node
cells are shown. It is apparent that the rate
acceleration induced by 0.3 µmol/l isoprenaline
involves a steeper slope of the diastolic
depolarization, with little or no change in other
action potential properties; similarly, a shallower
slope of the diastolic depolarization is the only
determinant of the rate slowing induced by
0.03 µmol/l acetylcholine (ACh).
The fact that neurotransmitter-induced rate
changes are concentrated on the pacemaker
depolarization implies that the mechanisms that
operate at low agonist concentrations are those
that specifically regulate phase 4 of the action
potential. This notion is in keeping with the
observation, originally reported in 1979,1 that If is
increased by adrenaline in a manner that is
compatible with adrenaline-induced rate
acceleration. The stimulatory action of betareceptor activation on If recorded in a sinoatrial
node is shown in Fig. 1b (left). Following this
initial finding, further investigation demonstrated
that beta-adrenergic stimulation increases If via a
shift in the current activation curve to more
positive voltages.12,13 An example of this effect is
shown in Fig. 1c (left). Clearly, during betareceptor stimulation a larger fraction of If current
is available at each voltage, which explains the
acceleration in rate of diastolic depolarization.
G22
The rightward shift in the If activation curve is
caused by an increase in the level of intracellular
cyclic adenosine monophosphate (cAMP) — the
second messenger that mediates If modulation.7
The increased rate of spontaneous activity
associated with moderate sympathetic activity can
therefore be explained by the following cascade of
cellular events: beta-receptor stimulation
activates adenylate cyclase and increases the
synthesis of cAMP, which binds to If channels and
shifts their activation curve to more positive
voltages, eventually leading to an increased
inward current and a steeper slope of diastolic
depolarization.
An opposite series of events regulates the rate
slowing caused by low concentrations of ACh.16 In
contrast to the hypothesis, long held by cardiac
physiologists, that the negative chronotropic
action of ACh was exclusively due to activation of
an ACh-dependent K+ current (IK,ACh), comparative
experiments in sinoatrial node cells demonstrated
the following:17 that low doses of ACh inhibit If,
and slow the rate, without affecting IK,ACh; and
that an increase in K+ conductance is only
obtained with much higher ACh concentrations.
The decrease in If involves reduced cAMP
production and a shift of the current activation
curve to more negative voltages.18 Figure 1b and c
(right) shows samples of the ACh-dependent
decrease in If during hyperpolarizing steps and of
the negative shift of the If activation curve.
The ACh-mediated If inhibition has special
physiological relevance because it is responsible
for the vagal regulation of basal cardiac rhythm;
additionally, as discussed below, this process is the
physiological equivalent of pharmacological
interventions aimed at reducing heart rate by
inhibition of the If current.
Heart rate reduction induced by If blockers
As mentioned above, the question of the relevance
of If to pacemaker depolarization has long been
discussed7,14 and has been addressed by specific
experimental protocols aimed at determining the
effect on rate of If changes (for example, see
DiFrancesco).19
Clearly, the evidence discussed above that
modulation of If by autonomic transmitters leads
to pacemaker rate modulation highlights the
essential role of If. More direct evidence of If
involvement can be gained from the use of agents
that specifically interact with If channels, such as
If channel blockers, by which a ‘pharmacological
dissection’ of the contribution of If to rate can in
theory be estimated. It is essential for complete
D. DiFrancesco
dissection that a highly specific If channel
blockade is induced.
Figure 2a shows the action of Cs+, a known
blocker of If, on the spontaneous action potentials
and on the fully activated I/V relation of If, as
measured in sinoatrial node cells. The If block is
voltage dependent and increases at more negative
voltages, causing a distinct outward rectifying
behaviour and a region of negative slope at
hyperpolarized voltages (Fig. 2a, bottom). These
features were incorporated into a model of If
channel block by Cs+, according to which external
Cs+ ions must enter the channel for a fraction
(about 70%) of the membrane electrical field
before binding to the blocking site.20 The voltage
dependence of If block thus reflects the voltage
dependence of Cs+ access to its binding site within
the channel pore. If current block occurs rapidly
upon hyperpolarization to negative voltages and is
as rapidly relieved on depolarization to positive
voltages, reflecting the fact that Cs+ ions are
‘pushed’ toward, or ‘pulled’ from, the binding
site, according to the voltage applied.
As shown in the top panel of Fig. 2a,
spontaneous activity is slowed but not halted by
Cs+ — an observation often used to refute the
relevance of If to pacemaker generation.22
However, it is apparent from the I/V curve
modification in the presence of Cs+ (bottom panel)
that even millimolar Cs+ concentrations do not
fully block If at diastolic voltages (23—38% in the
range —65 to —45 mV).7 Note also that Cs+ slightly
prolongs the action potential duration, possibly
reflecting a reduction in the delayed K+
conductance; this is not unexpected because Cs+ is
a known K+-channel blocker.23
Quite a different type of block is observed with
UL-FS49 (zatebradine) — a ‘rate-reducing’ agent
(Fig. 2b). If block by zatebradine is also voltage
dependent, but it has a voltage dependence that
is opposite to that of Cs+ block (i.e. it increases at
more positive potentials; bottom panel of Fig. 2b).
Analysis of the action of zatebradine21 indicates
that If block is exerted from the intracellular side
of the membrane, and that it occurs only when
channels are in the open state (‘open channel
blocker’). The voltage dependence of block can be
reproduced by assuming that zatebradine
molecules enter the pore from the intracellular
side for a fraction of the membrane electrical
field before binding to the blocking site.21 When
measured from the external membrane side, this
fraction is about 61%; this value is not too
dissimilar from that of Cs+ block, suggesting that
both agents may interfere with the ion flow
through If channels at similar positions in the pore.
If inhibition: a novel mechanism of action
(a)
(b)
200 ms
50 mV
G23
200 ms
50 mV
zatebradine 0.3 µM
Cs 2 mM
200
mV —100
Cs 5 mM
—50
mV —100
—50
pA zatebradine
3 µM
—200
pA
—1000
—400
Fig. 2 If blockers reduce the rate of diastolic depolarization.
(a) Cs+ (5 mmol/l) caused a voltage-dependent block of the
fully activated I/V relation of If, with the fractional block
increasing steeply with hyperpolarization (bottom). If block by
Cs+ (2 mmol/l) slowed pacemaker activity in another cell by
diminishing the slope of diastolic depolarization (top); however, a modest prolongation of action potential duration
reflected partial block of K+ currents. (b) Zatebradine
(3 µmol/l) — a rate-reducing agent — also blocked the fully
activated If in a voltage-dependent manner, but with fractional block decreasing with hyperpolarization (bottom). Low concentrations of zatebradine (0·3 µmol/l) were able to decrease
spontaneous rate by modifying only the diastolic depolarization
(top), indicating a more specific If blocking action than with
Cs+. Fully-activated I/V relations in (a) and in the control curve
in (b) were measured as previously published (see, for example, DiFrancesco).20 The I/V curve in the presence of zatebradine in (b) was calculated as previously detailed.21
Temperature was 35°C throughout.
As shown in the top panel of Fig. 2b, the rate
slowing induced by zatebradine 0.3 µmol/l is due
to a reduction in diastolic depolarization slope,
without changes in duration of action potential. An
effect exerted solely on phase 4 of the action
potential, although variable in degree of
specificity, is typical of rate-reducing agents (see
below) and confirms the existence of a direct
relationship between If inhibition and reduction in
spontaneous cardiac rate; this is in agreement
with the view that If activation during diastolic
depolarization is indeed the mechanism
responsible for initiation and control of heart beat.
Action of the rate-reducing agent
ivabradine
A recently developed substance, namely
Procoralan® (ivabradine; Servier, Neuilly-surSeine, France), exhibits a high degree of
specificity as an If-blocking molecule,24 and exerts
a correspondingly specific action as a ratereducing agent (i.e. lack of inotropic side effects
when tested in vivo,25,26 suggesting potentially
useful applications in clinical settings. Ivabradine
acts on If channels more selectively than does Cs+
and zatebradine; the half-block concentration of
ivabradine measured upon repetitive activation/
deactivation steps is 1.5 µmol/l,27 whereas that of
zatebradine is 80 µmol/l,28 and 5 mmol/l Cs+
blocks less than 38% of the current at voltages in
the range —65 to —45 mV.7
Detailed investigation of the If-blocking
properties of ivabradine shows that the drug
blocks from the inside24 and requires open
channels to access its blocking site, acting
therefore as an open channel blocker. This
property is illustrated in Fig. 3.
In Fig. 3a, ivabradine (3 µmol/l) was superfused
during an activation/deactivation protocol, in
which steps to —100/+5 mV were repetitively
applied every 6 s from a holding potential of
—35 mV; the If amplitude measured at —100 mV
underwent a slow, time-dependent inhibition
caused by channel blockade until after about
180 s, when it settled to a steady-state level
corresponding to approximately 75% reduction
(compare traces in control and after full blockade
in the upper panel). Note that the blocking action
was reversible and that the current recovered
toward its original amplitude following washout.
If, on the other hand, ivabradine was perfused
while holding the membrane at —35 mV (when If
channels are closed) for 100 s, no blockade
developed; only by reapplying the normal
activation/deactivation protocol was development
of blockade restored (Fig. 3b). This proves that
ivabradine can access its binding site and exert its
blocking action only when If channels are open.
Similar experiments show that not only block
onset but also removal of block requires open
channels (not shown). Thus, the action of
ivabradine is compatible with the idea that drug
molecules can be ‘trapped’, when bound, by the
channel voltage-dependent gates located at the
intracellular entrance of the pore. A similar result
was obtained with another rate-reducing agent,
namely ZD 7288, in studies performed in
heterologously expressed HCN channels.29
The steady-state blockade of If by ivabradine, as
measured in the fully activated I/V relation, has
some peculiar properties when compared with
other rate-reducing agents. In Fig. 4, the fully
activated I/V relation measured in a single cell in
control conditions (closed circles) and that
obtained by multiplying the control I/V curve by
the fractional steady-state block curve at 3 µmol/l
ivabradine (open circles) are shown. Clearly,
substantial block is exerted only at voltages in
which If is outward, whereas the inward part of
the I/V relation is little affected. These data
G24
D. DiFrancesco
2s
(a)
250
pA
0
pA
—100
1
s
0
*
500
0
pA/pF
1
2
3
s
0
—1000
1
1
ivabradine
3 µM
500
mV
3
—100
2
indicate that ivabradine blocks If channels
preferentially when the current flow across
channels is outward, whereas a very modest
inhibition occurs when the current is inward. The
block by ivabradine cannot therefore be simply
explained in terms of a voltage-dependent block
such as is proposed for zatebradine,21 or in terms
of a channel state-dependent block as proposed
for ZD 7288.29 It is instead consistent with the idea
that ivabradine competes with permeating ions at
one of their binding sites along the permeating
pathway in the channel pore.
Conclusion
The data discussed above show that If activation is
the process that is mainly responsible for
generation of diastolic depolarization. Control of
the diastolic depolarization rate, and hence of
cardiac chronotropy, by the autonomic nervous
system is mediated by changes in the degree of
activation of If via changes in cAMP levels. If If
activation is a determinant of diastolic
depolarization and of the frequency of
spontaneous activity, then modifications in If
should lead to parallel changes in heart rate.
Indeed the finding that ‘heart rate reducing’
agents — molecules that induce heart rate
reduction without inotropic side effects — act by
specific If blockade confirms the essential role of
If in pacemaking.
—50
0
50
—10
* ivabradine 3 µM
Fig. 3 Ivabradine is an open channel blocker. Activation/deactivation voltage step protocols (—100 mV/+5 mV) were applied
at a frequency of 1/6 Hz and the current amplitude was measured during hyperpolarization to —100 mV. (a) Time course of If
during perfusion with ivabradine 3 µmol/l, showing the timedependent current inhibition by the drug (bottom). Sample
traces in (1) control and (2) after steady-state block are plotted in the top panel. (b) In a different cell, the same protocol
was interrupted and the cell was held at —35 mV while perfusing ivabradine (3 µmol/l); the activation/deactivation protocol
was resumed after 100 s. Traces in (1) control, (2) just after the
100 s period at —35 mV and (3) after steady-state block are
shown in the top panel. Temperature was 32.5°C.
control
10
*
pA
2
—200
—300
2
20
2s
(b)
500
pA
—20
Fig. 4 Voltage dependence of If block by ivabradine 3 µmol/l.
The fully activated I/V relation of If was obtained in a sinoatrial node myocyte (filled circles) as previously described (see
DiFrancesco et al.12) and fitted to a second order polynomial
curve (full line). Fitted values were multiplied by mean fractional block values previously measured (not shown) at appropriate voltages to yield a fully activated I/V relation,
corresponding to steady-state block by ivabradine (open circles, line through points).
Procoralan (ivabradine), a recently developed
rate-reducing substance, inhibits If channels with
atypical blocking features, leading to a specific
use dependence. The selectivity (the interaction
with the If channel vs other channels) and
specificity (the current dependence vs voltage
dependence of If inhibition) characteristics of
Procoralan appear therefore to render that agent
potentially safe and effective in various
pathologies that require independent reduction in
heart rate.
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