Ivabradine is a âheart rate-reducingâ agent able to slow heart rate, without complicating side-effects. Its action results
from a selective and specific block of pacemaker f-channels of the ...cardiac sinoatrial node (SAN). Investigation has shown
that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The
constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels,
of which four isoforms (HCN1â4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study
we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed
in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 μ m for mHCN1 and 2.0 μ m for hHCN4. Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened
by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels
were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between
open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1
channels. In summary our results indicate that ivabradine is an âopen-channelâ blocker of hHCN4, and a âclosed-channelâ blocker
of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version
of a previously developed model of f-channel kinetics.
We used the Dynamic Clamp technique for i) comparative validation of conflicting computational models of the hyperpolarization-activated funny current, If, and ii) quantification of the role of If in ...mediating autonomic modulation of heart rate. Experimental protocols based on the injection of a real-time recalculated synthetic If current in sinoatrial rabbit cells were developed.
Preliminary results of experiments mimicking the autonomic modulation of If demonstrated the need for a customization procedure to compensate for cellular heterogeneity. For this reason, we used a cell-specific approach, scaling the maximal conductance of the injected current based on the cell's spontaneous firing rate. The pacemaking rate, which was significantly reduced after application of Ivabradine, was restored by the injection of synthetic current based on the Severi-DiFrancesco formulation, while the injection of synthetic current based on the Maltsev-Lakatta formulation did not produce any significant variation. A positive virtual shift of the If activation curve, mimicking the Isoprenaline effects, led to a significant increase in pacemaking rate (+17.3 ± 6.7%, p < 0.01), although of lower magnitude than that induced by real Isoprenaline (+45.0 ± 26.1%). Similarly, a negative virtual shift of the activation curve significantly lowered the pacemaking rate (−11.8 ± 1.9%, p < 0.001), as did the application of real Acetylcholine (−20.5 ± 5.1%).
The Dynamic Clamp approach, applied to the If study in cardiomyocytes for the first time and rate-adapted to manage intercellular variability, indicated that: i) the quantitative description of the If current in the Severi-DiFrancesco model accurately reproduces the effects of the real current on rabbit sinoatrial cell pacemaking rate and ii) a significant portion (50–60%) of the physiological autonomic rate modulation is due to the shift of the If activation curve.
Different cardiac stem/progenitor cells have been recently identified in the post-natal heart. We describe here the identification, clonal expansion and characterization of self-renewing progenitors ...that differ from those previously described for high spontaneous cardiac differentiation. Unique coexpression of endothelial and pericyte markers identify these cells as cardiac mesoangioblasts and allow prospective isolation and clonal expansion from the juvenile mouse ventricle. Cardiac mesoangioblasts express many cardiac transcription factors and spontaneously differentiate into beating cardiomyocytes that assemble mature sarcomeres and express typical cardiac ion channels. Cells similarly isolated from the atrium do not spontaneously differentiate. When injected into the ventricle after coronary artery ligation, cardiac mesoangioblasts efficiently generate new myocardium in the peripheral area of the necrotic zone, as they do when grafted in the embryonic chick heart. These data identify cardiac mesoangioblasts as committed progenitors, downstream of earlier stem/progenitor cells and suitable for the cell therapy of a subset of juvenile cardiac diseases.
The "funny" (pacemaker) current has unusual characteristics, including activation on hyperpolarization, permeability to K(+) and Na(+), modulation by internal cAMP, and a tiny, single-channel ...conductance. In cardiac cells and neurons, pacemaker channels control repetitive activity and excitability. The recent cloning of HCN subunits provides new insight into the molecular basis for the funny channel properties.
The effects of ZD 7288, a "bradycardic" agent, in young rat hippocampal slices in vitro were studied. ZD 7288 (1-1000 microM) reduced the hyperpolarization-activated current (Ih) in CA1 pyramidal ...neurons by a voltage-independent blocking mechanism. Under current-clamp conditions, the bradycardic agent (10 microM) caused membrane hyperpolarization (by 5.9 +/- 0.5 mV) and a reduction of membrane conductance (by 17.9 +/- 4.1%). These data are consistent with the block of an inward current which is active at rest. The drug-induced hyperpolarization depressed the cell's excitability by increasing the threshold current necessary to induce firing. When the drug-induced hyperpolarization was compensated for by injection of a tonic depolarizing current, ZD 7288 caused a reduction of the inhibitory post-synaptic potential (IPSP) in EPSP-IPSP sequences. Since Cs+, another known blocker of Ih, is able to reverse long-term depression (LTD) of the CA3-CA1 synapse in hippocampal slices, we tested the effect of ZD 7288 on synaptic transmission. We found that ZD 7288 did not significantly modify LTD, suggesting that Cs+-induced inhibition of LTD maintenance is not directly related to block of Ih.
The ‘funny’ current, first described in cardiac pacemaker cells almost 30 years ago, is a key player in the generation of pacemaker activity and the autonomic modulation of heart rate. Because of ...these specific functions, a search for molecules able to interfere selectively with the ‘funny’ current was undertaken soon after its discovery, with the aim of developing tools for the pharmacological control of heart rate. This search has succeeded in generating a new class of drugs, the heart rate-reducing agents, which act through specific blockade of f-channels; one of these drugs, ivabradine, is presently marketed against stable angina. Because of their many functions in heart and other tissues, pharmacological utilization of “funny” channel properties is an exciting new frontier open to further developments.
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.
Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require ...closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.
Serious workings of the funny current DiFrancesco, Dario
Progress in biophysics and molecular biology,
2006, 2006 Jan-Apr, 2006-01-00, 20060101, Letnik:
90, Številka:
1
Journal Article
Recenzirano
Since its first description in 1979 (Brown et al., 1979. Nature 280, 235–236), extensive work on the
I
f current has amply demonstrated its role in the generation and neurotransmitter-induced ...modulation of pacemaker activity in heart (DiFrancesco, 1993. Annual Review of Physiology 55, 455–472). At pacemaker voltages,
I
f is an inward current activated by negative voltage and by intracellular cAMP. Moderate
β
-receptor stimulation accelerates, and vagal stimulation slows, cardiac rate by increasing and decreasing, respectively,
I
f at diastolic potentials via changes in cAMP level. Cloning of four isoforms of hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels in the late 1990s has shown their correlation to native f-channels. Comparison of the properties of native pacemaker channels with those of HCN channels has provided information concerning the composition and molecular features of native channels in different cardiac regions. The relevance of
I
f to pacemaker generation and modulation makes f-channels a natural target of drugs aiming to control pharmacologically heart rate. Agents selectively reducing heart rate have been developed which act by specific inhibition of
I
f, such as ivabradine; these drugs have a high potential for treatment of diseases where heart rate reduction is beneficial, such as angina and heart failure. Knowledge of the molecular properties of HCN clones will help the development of drugs specifically interacting with cardiac, rather than neuronal pacemaker channels. Devices able to replace electronic pacemakers and based on the delivery of a cellular source of pacemaker channels to non-pacing tissue (biological pacemakers) are likely to be developed in the near future for use in therapies for diseases of heart rhythm.
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