Download Pacemaker current (If) in the human sinoatrial node

Clinical research
European Heart Journal (2007) 28, 2472–2478
doi:10.1093/eurheartj/ehm339
Arrhythmia/electrophysiology
Pacemaker current (If) in the human sinoatrial node
Arie O. Verkerk1, Ronald Wilders1, Marcel M.G.J. van Borren1, Ron J.G. Peters2, Eli Broekhuis3,
Kayan Lam3, Ruben Coronel1, Jacques M.T. de Bakker1,4, and Hanno L. Tan1,2*
1
Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;
Department of Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 3Department
of Cardiothoracic Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; and
4
Interuniversity Cardiology Institute the Netherlands, Utrecht, The Netherlands
2
Received 15 February 2007; revised 1 June 2007; accepted 17 July 2007; online publish-ahead-of-print 6 September 2007
KEYWORDS
Electrophysiology;
Ion channels;
Pacing;
Sinoatrial node;
Action potentials
Aims Animal studies revealed that the hyperpolarization-activated pacemaker current, If, contributes to
action potential (AP) generation in sinoatrial node (SAN) and significantly determines heart rate. If is
becoming a novel therapy target to modulate heart rate. Yet, no studies have demonstrated that If is
functionally present and contributes to pacemaking in human SAN. We aimed to study If properties in
human SAN.
Methods and results In a patient undergoing SAN excision, we identified SAN using epicardial activation
mapping. From here, we isolated myocytes and recorded APs and If using patch-clamp techniques. Pacemaker cells generated spontaneous APs (cycle length 828 + 15 ms) following slow diastolic depolarization,
maximal diastolic potential 261.7 + 4.3 mV, and maximal AP upstroke velocity 4.6 + 1.2 V/s. They exhibited an hyperpolarization-activated inward current, blocked by external Csþ (2 mmol/L), characterizing it as
If. Fully-activated conductance was 75.2 + 3.8 pS/pF, reversal potential 222.1 + 2.4 mV, and half-maximal
activation voltage and slope factor of steady-state activation 296.9 + 2.7 and 28.8 + 0.5 mV. Activation
time constant ranged from 350 ms (2130 mV) to 1 s (2100 mV), deactivation time constant 156 +
45 ms (240 mV). The role of If in pacemaker activity was demonstrated by slowing of pacemaker cell diastolic depolarization and beating rate by Csþ.
Conclusion If is functionally expressed in human SAN and probably contributes to pacemaking in human SAN.
Introduction
Cardiac pacemaking is a basic physiological function
required to match cardiac performance to metabolic
demand. In the mammalian heart, it is accomplished by
pacemaker cells in the sinoatrial node (SAN) region. The
SAN is a complex tissue with regional differences in morphological and electrical properties.1 Animal studies have
revealed that pacemaking in SAN cells follows from diastolic
depolarization driven by a net inward current, which results
from an interaction of multiple ion currents.1,2 Inward
currents are activated during diastole: hyperpolarizationactivated pacemaker current (If),3,4 background Naþ
current (Ib,Na),5 sustained inward current (Ist),6,7 T- and
L-type Ca2þ currents (ICa,T and ICa,L, respectively),8 and
Ca2þ-release activated Naþ–Ca2þ exchange current (INCX).9
Conversely, outward currents are deactivated: rapid
delayed rectifier Kþ current (IKr) and slow delayed rectifier
Kþ current (IKs).10–13 The relative contributions of these
* Corresponding author: Heart Failure Research Center, Department of
Clinical and Experimental Cardiology, Academic Medical Center, Meibergdreef
9, 1105 AZ Amsterdam, The Netherlands. Tel: þ31 20 5663264; fax: þ31 20
6975458.
E-mail address: [email protected]
currents to diastolic depolarization are a matter of
debate.14–16 Still, If consistently exhibits a key role in
animal studies, mediating the heart-rate modulating
actions of autonomic neurotransmitters,17 and underlying
heart-rate slowing by aging18 and heart failure.19,20 Human
studies also show, albeit indirectly, that If is clinically relevant. Recent studies linked familial SAN disease to
mutations in HCN4,21–23 a molecular component of If.24
Accordingly, If is evolving into a novel therapy target: If
blocking drugs are being developed to lower heart rates
and be beneficial in ischaemic heart disease,25 while If
gene transfer may relieve disease-causing bradycardias.26,27
Yet, direct evidence that If is functionally present in the
human SAN is still lacking, as the cellular electrophysiological properties of human SAN are virtually unexplored, given
the extreme difficulty of obtaining human SAN preparations
for in vitro electrophysiological studies. To date, the only
study on adult human SAN utilized explanted hearts of
patients in end-stage heart failure who underwent cardiac
transplantation.28 However, that study was limited
because, in addition to possible confounding effects of endstage heart failure, ion currents underlying SAN action
potentials (APs) were not studied. In the present study, we
explored the electrophysiological properties of isolated
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007.
For permissions please email: [email protected].
Pacemaker current in human sinoatrial node
SAN cells of a patient without structural heart disease who
underwent SAN excision, in particular, AP properties and
the presence and functional role of If.
Methods
This study complied with institutional guidelines. The patient
provided written informed consent.
Epicardial mapping
We used a flexible plaque electrode harbouring 64 terminals
arranged in an 8-by-8 matrix at inter-electrode distances of
6.5 mm. Recordings were made with a 256-channel mapping
system (BioSemi ActiveTwo, 24-bit resolution) in unipolar mode.
An electrode attached to the mediastinum served as reference.
Cell isolation
The excised tissue was immediately submerged in Tyrode’s solution
(48C) and cell isolation was started. The region including and surrounding the origin of the spontaneous rhythm during surgery was
cut into cubic chunks (,1 mm3), and single cells were enzymatically
isolated as described previously.29
Cellular electrophysiology
Cell suspensions were placed in a recording chamber on the stage of
an inverted microscope, and superfused with Tyrode’s solution,
which contained 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.5
glucose, 5.0 HEPES (pH 7.4 with NaOH). Membrane potentials and
currents were recorded at 36 + 0.28C in the whole-cell configuration of the patch-clamp technique. The pipette solution contained
(mmol/L): 125 K-gluconate, 20 KCl, 5.0 NaCl, 1.0 MgCl2, 5.0 MgATP,
10.0 HEPES (pH 7.2 with KOH). Potentials were corrected for liquid
junction potential, and membrane capacitance (Cm) and series
resistance (Rs) were compensated for .85%. Signals were low-pass
filtered (cut-off frequency 5 kHz) and digitized at 10 kHz. If was
assessed as the Csþ-sensitive, time-dependent current that activated during 2 s hyperpolarizing voltage-clamp steps from a
holding potential of 240 mV (step interval: 4 s). The If activation
curve was obtained by plotting normalized Csþ-sensitive tail
current amplitude against potential, and characterized by fitting
it to the Boltzmann equation I/Imax ¼ A/{1.0þexp[(V2V1/2)/k]} to
determine the membrane potential for half-maximal activation
(V1/2) and the slope factor (k). The time courses of Csþ-sensitive If
activation and deactivation were fitted by the monoexponential
equations I/Imax ¼ A[12exp(2t/t)] and I/Imax ¼ Aexp(2t/t),
respectively, ignoring the variable initial delay in If activation.4
Statistics
Results are expressed as mean + SEM. AP parameters from 10 consecutive APs were averaged. Two sets of data were considered
significantly different if the probability value of the unpaired Student’s t-test was ,0.05.
Results
Patient
A 49-year-old woman had paroxysmal tachycardias from the
age of 28 years, which were usually regular and developed
within minutes. There was no family history of cardiac
arrhythmias and no signs of structural heart disease, as
repeated echocardiography, coronary angiography, and invasive hemodynamic studies were normal. Holter recordings
and ECGs revealed various tachyarrhythmias, notably atrial
2473
fibrillation, and different regular narrow-complex tachycardias with antegrade P waves (RP.PR) whose morphology
was either similar or dissimilar to sinus rhythm (120–150/
min). There was an almost permanently enhanced sinus
rate (average daytime rate 100–120/min, average nighttime rate 90/min), suggestive of inappropriate sinus tachycardia (IST) (Figure 1A). Invasive electrophysiological
studies showed no evidence of accessory pathways or dual
AV nodal physiology. Atrial tachycardias were noninducible
upon programmed stimulation, burst pacing, and isoproterenol or acetylcholine infusion, precluding any opportunity
for RF catheter ablation. Thus, various antiarrhythmic
drugs were used, including pindolol, metoprolol, verapamil,
flecainide, propafenone, and amiodarone. As these drugs
were either ineffective or, in the case of b-blockers, not tolerated because of side effects, a DDD pacemaker with supraventricular antitachycardia pacing algorithms (Medtronic
AT500) was implanted. However, antitachycardia pacing
did not terminate the supraventricular arrhythmias, and
the patient did not tolerate the frequent antitachycardia
pacing episodes. After repeated burst pacing attempts to
induce permanent atrial fibrillation had failed, His bundle
ablation was performed. Yet, the supraventricular tachyarrhythmias caused frequent mode switches to VVI pacing
that resulted in pacemaker syndrome, which the patient tolerated poorly. It became increasingly clear that the patient
suffered severely from the IST episodes (palpitations both at
rest and already at minimal levels of exercise). We found no
evidence for extrinsic dysregulation in the vagosympathetic
balance, as autonomic function tests were normal
(responses upon respiratory, Valsalva, and orthostatic
manoeuvres). Thus, we directed our efforts at relieving
these IST episodes by the use of the putative If blocker ivabradine (10 mg bid).25 This drug initially slowed baseline
heart rates and partly suppressed tachycardia episodes.
However, during prolonged use, these effects waned. Subsequently, we performed two procedures to extinguish the
SAN using endocardial RF catheter ablation, but were unsuccessful, presumably because of the subepicardial localization of the SAN.30 Finally, we conducted surgical SAN
excision.
SAN excision and postoperative follow-up
The patient had discontinued all antiarrhythmic drugs for at
least three half-lives before SAN excision (including amiodarone and ivabradine, which were discontinued 11
months and 8 weeks before surgery, respectively). At the
onset of extracorporeal circulation, she had sinus rhythm
(cycle length 655 ms) as assessed by ECG. Intraoperative
epicardial mapping revealed earliest activation at the
anterior aspect of the superior caval vein, 1 cm cranial
from the right atrium, followed by centrifugal spread of
activation (Figure 2A and B). After marking the point of earliest activation with a felt-tip pen, we resected this region
and closed the defect with an autologous pericardial patch.
During a 30 months’ postoperative follow-up, there was
no more documentation of sinus rhythm. The patient had
atrial rhythm (Figure 1B) or AV sequentially paced rhythm
(not shown). She had no more sinus tachycardia episodes.
However, she did remain symptomatic of atrial fibrillation/
flutter.
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A.O. Verkerk et al.
Figure 1 (A) Inappropriate (at rest) sinus tachycardia episode before sinoatrial node excision. (B) Atrial rhythm with ventricular pacing at postoperative
follow-up. Note that the P-wave shape and axis are clearly different from the sinus tachycardia episode in (A)
Morphological and electrophysiological properties
of single cells of the SAN region
We performed enzymatic isolation of cells from the resected
tissue (region of earliest activation) for single-cell studies.
Consistent with previous reports, the living-cell yield was
low (,5%). Pacemaker cells and atrial myocytes exhibited
distinct morphologies and AP configurations (Figure 2C and
D; Table 1).4 Pacemaker cells had an irregular, bent,
spindle shape and a paucity of myofilaments, along with
spontaneous contractions and rhythmic pacemaker activity
Pacemaker current in human sinoatrial node
2475
diastolic threshold. Most AP parameters differed significantly between both cell types. Atrial myocytes had a
stable resting membrane potential, whereas pacemaker
cells had a less negative maximum diastolic potential
(MDP) and diastolic depolarization, which resulted in pacemaker activity. The maximal AP upstroke velocity (dV/
dtmax) was typically slower in pacemaker cells than in
atrial myocytes. In both cell types, APs overshot the zero
potential value, but the overshoot was higher in atrial myocytes. APs of atrial myocytes repolarized earlier and faster,
resulting in shorter AP duration at 20% (APD20) and 50%
(APD50), but not 90% (APD90) of repolarization. The If
blocker Csþ (2 mmol/L) slowed pacemaker cell activity
(26% cycle length increase) by slowing diastolic depolarization (Figure 2E), leaving AP overshoot and duration
unaltered.
If properties in pacemaker cells
Figure 2 (A) Mapping electrode array over right atrium (RA) containing 64
electrodes (dots) in an 8 8 matrix, positioned 6.5 mm apart. RV, right ventricle; RAA, right atrial auricle; LA, left atrium; LAD, left anterior descending
artery; SVC, superior caval vein; IVC, inferior caval vein; PA, pulmonary
artery. (B) Activation map obtained with mapping electrode. Colours indicate
moment of local activation (inset); lines are 10 ms isochrones. (C, D) Photographs (top) and action potentials (bottom) of an isolated pacemaker cell
(spontaneous electrical activity, C) and atrial myocyte (stimulated at 2 Hz,
D). (E) Effect of Csþ on the action potential of a single pacemaker cell.
(Figure 2C). Atrial myocytes were rod-shaped with a clear
striation pattern (Figure 2D) and were significantly longer
and wider than pacemaker cells; consequently, membrane
capacitance (Cm) was larger (Table 1).
Atrial myocytes were quiescent or exhibited irregular slow
spontaneous contractions due to spontaneous Ca2þ release
(intracellular Ca2þ was measured using Indo-1, data not
shown). Spontaneously contracting atrial myocytes are a
consistent finding in human atrial-cell isolations.31,32 In the
present study, the irregularly beating atrial myocytes had
resting membrane potentials between 210 and 240 mV.
Typically, these myocytes went into contracture within
15 min after the start of superfusion and were excluded
from further analysis.
Figure 2C and D shows typical APs of a pacemaker cell and
an atrial myocyte (note different time scales); average AP
properties are summarized in Table 1. APs of atrial myocytes
were elicited at 2 Hz by 3 ms current pulses 50% above
Subsequent voltage-clamp experiments revealed that pacemaker cells had an inward current that activated following
2 s hyperpolarizing steps from 240 mV (Figure 3A). This
hyperpolarization-activated current became larger and activated more rapidly at increasingly negative potentials.
Moreover, it was strongly reduced by 2 mmol/L Csþ
(Figure 3A, middle), characterizing it as If.4,33 Its properties
were analysed from the Csþ-sensitive current (Figure 3A,
bottom). Average maximal If conductance was 75.2 +
3.8 pS/pF (Figure 3B) and the reversal potential was
222.1 + 2.4 mV, as determined from comparison of the
fully-activated current (Istep) at 2130 mV and the associated
tail current (Itail) at 240 mV. Voltage-dependence of activation was analysed by plotting normalized Itail amplitude
against the preceding voltage steps. Activation threshold
was between 250 and 260 mV (Figure 3C), and the
average half-maximal activation voltage (V1/2) and slope
factor of the Boltzmann fit to the data were 296.9 + 2.7
and 28.8 + 0.5 mV, respectively (Figure 3C). Activation
and deactivation time constants were obtained from monoexponential fits of the step and tail currents, respectively.
Activation time constants ranged from 350 ms at
2130 mV to 1 s at 2100 mV, and deactivation time
constant was 156 + 45 ms at 240 mV (Figure 3B).
Discussion
We provide the first demonstration that human SAN pacemaker cells functionally express If. If probably contributes
to diastolic depolarization and pacemaking in human SAN,
as evidenced by the effects of Csþ to block If and reduce
pacemaker cell beating rates by slowing diastolic depolarization. Yet, in accordance with animal studies,4 Csþ did not
abolish pacemaker activity completely, indicating that
other ion currents are also involved in cardiac pacemaking.
Compensatory mechanisms may attenuate the effects of If
blockade. For instance, removal of depolarizing current
carried by If hyperpolarizes maximum diastolic potential4
and slows diastolic depolarization rate, thereby increasing
other inward currents (e.g. the background Naþ current34)
and decreasing outward currents (potassium current is
reduced because potassium driving force is reduced and
deactivation rate is increased).
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A.O. Verkerk et al.
Table 1 General properties of single human pacemaker and atrial cells
Pacemaker cells
Morphology
Cell length (mm)
Cell width (mm)
Electrophysiology
Cm (pF)
Cycle length (ms)
MDP (mV)
DDR100 (mV/s)
dV/dtmax (V/s)
APA (mV)
Overshoot (mV)
APD20 (ms)
APD50 (ms)
APD90 (ms)
66.7 + 6.3 (n ¼ 4)
7.8 + 0.4 (n ¼ 4)
56.6 + 8.7 (n ¼ 4)
828 + 15 (n ¼ 3)
261.7 + 4.3 (n ¼ 3)
48.9 + 18 (n ¼ 3)
4.6 + 1.2 (n ¼ 3)
78.0 + 4.5 (n ¼ 3)
16.4 + 0.7 (n ¼ 3)
64.9 + 16.9 (n ¼ 3)
101.5 + 27.0 (n ¼ 3)
143.5 + 34.9 (n ¼ 3)
Atrial cells
87.8 + 4.9 (n ¼ 4)*
16.7 + 0.9 (n ¼ 4)*
88.3 + 10.7 (n ¼ 5)*
—
279.7 + 4.1 mV (n ¼ 3)*
—
243 + 96 (n ¼ 3)*
124.2 + 8.0 (n ¼ 3)*
44.5 + 4.3 (n ¼ 3)*
3.1 + 1.1 (n ¼ 3)*
11.6 + 4.7 (n ¼ 3)*
92.3 + 28.2 (n ¼ 3)
Data are mean + SEM; n, number of cells; Cm, membrane capacitance; MDP, maximal diastolic potential; DDR100, diastolic
depolarization rate over the 100 ms time interval starting at MDP; dV/dtmax, maximal upstroke velocity; APA, action potential
amplitude; APD20, APD50, and APD90, action potential duration at 20, 50, and 90% repolarization. *P , 0.05.
Figure 3 Voltage- and time-dependence of the hyperpolarization-activated inward current, If, in single pacemaker cells. (A) Top: superimposed original current
recordings of If elicited by various voltage steps (voltage-clamp protocol in inset). Middle: effects of external Csþ on If. Bottom: Csþ-sensitive current obtained by
digital subtraction of current traces recorded in the absence and presence of external Csþ. Arrows indicate If step (Istep) and tail (Itail) current. (B) Average
current–voltage relationship of the If step and tail currents (n ¼ 3). (C ) Steady-state activation curve (dashed line is Boltzmann fit to the experimental data)
and time constants of (de)activation (n ¼ 3).
The If activation threshold (between 260 and 250 mV)
and V1/2 (296.9 + 2.7 mV) were at more hyperpolarized
potentials than in a recent study in which wild-type HCN4
was heterologously expressed.35 These differences may be
partly explained by the different experimental models.
Moreover, our hyperpolarizing shift of If activation may be
due to (1) absence of cyclic AMP from the pipette solution,36
(2) cell dialysis,35 and (3) lack of full activation of If during
the 2 s voltage-clamp steps at less negative potentials. Of
note, the AP properties of the single pacemaker cells
studied here differed markedly from those reported previously in a study of intact human SAN preparations.28
Most notably, the single cells in our study had faster spontaneous beating rates (cycle lengths 828 vs. 2300 ms). It
must be noted, however, that the reported intact SAN preparations were obtained from explanted hearts of patients
Pacemaker current in human sinoatrial node
in end-stage heart failure,28 although heart failure was not
present in our study. It is likely that these differences in
AP properties are better explained by the effects of heart
failure than by the type of preparation (intact node vs. isolated cells), as intrinsic SAN rate in humans is reduced in
heart failure.37 Moreover, in a rabbit model of heart
failure, the intrinsic rates of single SAN cells19 and intact
SAN preparations38 were reduced to the same extent.
Molecular characterization of If has pointed to the
hyperpolarization-activated cyclic nucleotide (HCN) gated
family, which comprises four members, HCN1–4.24 All
except HCN3 are present in the heart, with their expression
varying somewhat among species, cardiac tissue, and age.39
In SAN of various species, HCN4 is the dominant HCN
isoform, constituting 80% of the total HCN message.40,41
Our demonstration of If in human SAN pacemaker cells
agrees with previously reported links between familial SAN
disease and HCN4 mutations.21–23 We did not attempt molecular characterization of SAN pacemaker cells, because
we allocated the entire SAN region for cell isolation and
chose to focus on obtaining as many electrophysiological
data as possible. At the same time, we expected that the
yield of enzymatic dissociation would be low and sufficient
only for cellular electrophysiological studies.
Study limitations
This study is based on one patient, whose SAN was excised
because of tachycardias which were resistant to drug
therapy and RF catheter ablation. We cannot rule out that
the SAN pacemaker cell If properties found here are not
fully representative of the general population. Yet, obtaining human SAN cells for patch-clamp studies is extremely
difficult, as SAN excision should be conducted only in
patients who are refractory to all conventional therapies,
as demonstrated in the present case. Similarly, it is unlikely
that SAN excision of healthy subjects will ever be conducted, and large-scale studies of fully normal human SAN
cells are inconceivable.
Conclusion
Human SAN cells have pacemaker activity qualitatively
similar to that of various species. If probably contributes
to pacemaking in the human SAN. These findings provide
support for novel If targeting therapies, using new drug
classes or gene transfer, aimed at common diseases, such
as ischaemic heart disease and sick sinus syndrome, to
reduce or increase heart rate.
Acknowledgements
The authors thank Jan Zegers and Jan Bourier for excellent technical assistance.
Conflict of interest: none declared.
Funding
H.L.T. was supported by the Royal Netherlands Academy of Arts and
Sciences (KNAW), the Netherlands Heart Foundation (NHS2002B191,
NHS2005B180), and the Bekales Foundation.
2477
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