Download The slow mo mutation reduces pacemaker current and heart rate in

Am J Physiol Heart Circ Physiol
281: H1711–H1719, 2001.
The slow mo mutation reduces pacemaker
current and heart rate in adult zebrafish
KERRI S. WARREN,1* KEITH BAKER,2* AND MARK C. FISHMAN1
1
Cardiovascular Research Center and 2Department of Anesthesia and Critical Care, Massachusetts
General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129
Received 9 February 2001; accepted in final form 3 July 2001
bradycardia; genetic screen; Ih
provide a means to scan
the genome for genes important to embryonic physiological function (35, 36). However, many important
homeostatic mechanisms are not fully developed until
well after embryogenesis, and it is not known if the
functions of a gene in the embryo predict those in the
adult. The regulation of heart rate, for example, differs
dramatically from embryonic to adult animals (4, 5, 18,
30). Neuronal and humoral control and developmentally regulated and chamber-specific participation of
voltage-gated currents are all core determinants of
adult cardiac pacing. Additional factors that change
with growth and that may affect heart rate include
oxygen delivery to the thickening myocardium, calcium
storage and release mechanisms, sarcomeric kinetics,
the rate of force development in cardiomyocytes, and
properties of the pacemaker potential (19).
How these many heart rate regulators are orchestrated to provide carefully controlled sequential beatGENETIC SCREENS IN ZEBRAFISH
* Authors contributed equally to this study.
Address for reprint requests and other correspondence: M. C.
Fishman, Cardiovascular Research Center, Massachusetts General
Hospital, 149 13th St., Charlestown, MA 02129 (E-mail: fishman
@cvrc.mgh.harvard.edu).
http://www.ajpheart.org
ing of the chambers is not clear. Many heart cells
appear to have pacemaking properties when isolated in
culture. The “pacemaker current” (Ih) is one current
shared among cardiac myocytes, both in the sinus node
and throughout atria and ventricles. There are several
lines of evidence that Ih is among the most important
currents for pacemaking in the heart (for a review, see
Ref. 11). It is an unusual current in that it provides
inward current upon hyperpolarization of the cell
membrane. The inward current helps lead to sufficient
depolarization to initiate an action potential. Ih is a
presumptive target for autonomic nervous system regulation of heart rate. Adrenergic stimulation increases
Ih activity and thereby speeds heart rate. Cholinergic
stimulation decreases Ih activity and thereby slows
heart rate (9). Ih properties differ between the embryonic and adult heart and between the atrium and
ventricle. As the heart matures, Ih conductance in the
atrium and the developing conduction system increases, whereas the current density of Ih in the ventricle drops dramatically (6). In addition, the threshold
for activation of ventricular Ih is shifted to an even
more hyperpolarized voltage (27, 29), thus reducing its
effect on pacemaking.
The genetic evidence that Ih does regulate cardiac
pacemaking in the intact animal comes from the mutation slow mo. We discovered this mutation as part of
a large-scale zebrafish genetic screen. The homozygous
slow mo fish has a slow heart rate (sinus bradycardia)
evident from the first heartbeats (3). This is the only
noticeable effect of the mutation. At present, the mutation resides in an unknown gene. In a prior study, we
developed the means to culture and voltage-clamp cells
from embryonic zebrafish hearts and found these cells
manifested a variety of currents, including Ih. In the
embryonic zebrafish heart, Ih appears to have two
kinetically distinct components, as has also been described in cardiomyocytes from the rabbit heart (10, 20,
24) and in neurons from the rat (31). Myocardial cells
of slow mo mutant embryos have a markedly reduced
Ih, with a near ablation of the fast component (3).
Most of the 150 cardiac mutations discovered in
zebrafish genetic screens are lethal (2, 7, 32, 36). AlThe costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
H1711
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Warren, Kerri S., Keith Baker, and Mark C. Fishman.
The slow mo mutation reduces pacemaker current and heart
rate in adult zebrafish. Am J Physiol Heart Circ Physiol 281:
H1711–H1719, 2001.—Genetic studies in zebrafish have focused on embryonic mutations, but many physiological mechanisms continue to mature after embryogenesis. We report
here that zebrafish homozygous for the mutation slow mo can
be raised to adulthood. In the embryo, the slow mo gene is
needed to regulate heart rate, and its mutation causes a
reduction in pacemaker current (Ih) and slowing of heart rate
(bradycardia). The homozygous adult slow mo fish continues
to manifest bradycardia, without other evident ill effects.
Patch-clamp analysis of isolated adult cardiomyocytes reveals that Ih has chamber-specific properties such that the
atrial current density of Ih is far greater than the ventricular
current density of Ih. Ih is markedly diminished in cardiomyocytes from both chambers of slow mo mutant fish. Thus Ih
continues to be a critical determinant of pacemaker rate even
after adult neural and humoral influences have developed. It
is clear that zebrafish may be used for genetic dissection of
selected physiological mechanisms in the adult.
H1712
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
though slow mo mutant embryos do have reduced viability, we have been able to carry several through to
adulthood. In the adult fish, we found that the slow mo
mutation causes a chronic bradycardia even though
the heart is fully innervated. Here, we report maturational changes in the properties of Ih that have a
chamber-specific profile. The adult slow mo mutant
fish continues to manifest bradycardia. This bradycardia is present in the setting of a dramatic reduction in
Ih, especially in atrial cells. This demonstrates that Ih
continues to play a central role in pacemaking in the
adult heart.
MATERIALS AND METHODS
AJP-Heart Circ Physiol • VOL
RESULTS
Adult slow mo zebrafish have a reduced heart rate. Of
the nearly 150 published mutations affecting the heart
in zebrafish, only slow mo homozygotes are viable past
embryonic development. Slow mo fish grow at the same
rate as wild-type siblings but do appear to have a
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Fish care. Zebrafish were raised and maintained as described in (37). Fish were classified as adults after achievement of sexual maturity at ⬃3 mo of age. Homozygote genotypes for slow mo mutant adults were confirmed before
analysis.
Heart rate counts. Adult fish (4–18 mo old) were transferred to an aqueous solution of propofol at a final concentration of 5 ␮g/ml. Fish were anesthetized for 5 min and then
transferred to the counting chamber. The chamber consisted
of a cuvette immersed in propofol at the same concentration
used in the anesthetizing container. The chamber made it
possible to turn the fish ventral side up to directly observe
the heart beat. The heart rate was counted for a full 60 s
under direct vision using a dissecting microscope. Once the
counting was finished, the fish was transferred to normal fish
water without propofol. All fish survived the counting experiments. Temperature of all solutions used for the propofol
counting experiments fell within the range of 20.0–23.0°C. A
second set of counting experiments was conducted with the
fish anesthetic tricaine (MS-222, Sigma) at a final concentration of 0.16 mg/ml (37), and the temperature was maintained
at 26°C throughout. Heart rates were always determined
blind to the genotype of the fish.
Cardiomyocyte culture. Cells were prepared from adult
zebrafish (4–18 mo old). Fish were anesthetized by allowing
the fish to swim in an aqueous solution of tricaine for 3–5
min. Each fish was in turn placed in L15 media (GIBCOBRL) and pinned ventral side up, and the entire heart was
dissected out and placed in a solution of L15 supplemented
with penicillin G (200 IU/ml) and streptomycin (200 ␮g/ml).
The heart could reliably be removed within 30 s. Once three
hearts had been placed in the antibiotic supplemented L15,
the atrium and ventricle were separated under direct vision.
The chambers of interest (either atria or ventricles) were
then opened widely and transferred to 750 ␮l of solution A
[containing (in mM) 135 NaCl, 5.4 KCl, 1 MgCl2, and 10
HEPES (pH 7.20 with NaOH)]. The solution was supplemented with penicillin G-streptomycin (26). The chambers
were allowed to stir for 10 min at room temperature in a
small cuvette. While the hearts continued to stir, 750 ␮l of
solution B (solution A supplemented with 2 mg/ml collagenase I, 0.28 mg/ml protease XIV, 200 IU/ml penicillin G, and
200 ␮g/ml streptomycin) was added. The hearts were then
allowed to stir for 60 min at room temperature to digest the
tissue. The digestion solution was then transferred to an
Eppendorf tube, and the cells were pelleted by spinning at
2,000 rpm for 2 min. The supernatant was removed, and 1 ml
of L15 supplemented with penicillin G-streptomycin was
added. The cells were spun as before, and the supernatent
was again removed. The cells were then resuspended in 400
␮l of L15 supplemented with penicillin G-streptomycin. The
cells were rested for 10 min at room temperature, and single
cells were then obtained by gentle trituration. Cells were
placed on plain glass coverslips. The cells were used for
recording on the following day. The cells were prepared blind
to the genotype of the fish.
Electrophysiology. Standard whole cell recording techniques were applied to single cells. All data were acquired
and analyzed blind to genotype. For recording Ih from both
atria and ventricles, the bath solution contained (in mM) 40
NaCl, 40 KCl, 2 CaCl2, 1 MgCl2, 40 N-methyl-D-glucamine,
10 tetraethylammonium chloride, 10 HEPES, and 10 glucose
(pH 7.4 with NaOH). The corresponding pipette solution
contained (in mM) 70 KF, 70 KCl, 1 MgCl2, 10 EGTA, and 10
HEPES (pH 7.4 with NaOH). For recording sodium current
(INa) from both atria and ventricles, the bath solution contained (in mM) 30 NaCl, 110 choline chloride, 2 KCl, 1 MgCl2,
2 CaCl2, 2 BaCl2, 10 HEPES, and 10 glucose (pH 7.4 with
NaOH). The corresponding pipette solution contained (in
mM) 70 CsCl, 70 CsF, 0.5 MgCl2, 10 EGTA, and 10 HEPES
(pH 7.4 with CsOH). Pipettes were pulled using borosilicate
glass and polished to resistances of 1.6–3.2 M⍀. All INa
recordings had series resistance compensation employed at
90%. Ih were acquired without such compensation. Data
acquisition and analysis were carried out using pCLAMP 6
software (Axon Instruments; Foster City, CA). All recordings
were at room temperature (20.5–23.0°C). The amplitude of Ih
was determined from the amplitude of the Boltzmann fit to
the steady-state activation curve determined at a test voltage
of ⫺130 mV. The amplitude of INa was determined from the
amplitude of the Boltzmann fit to the steady-state inactivation curve determined at a test voltage of ⫺30 mV. The leak
current and nonlinear open channel properties were minimized by taking all of the current amplitudes at a constant
test voltage. The kinetics of activation for Ih were determined
at ⫺130 mV. Data traces were filtered at 1 kHz and fitted
with either a single- or double-exponential curve. All traces
were initially fit with a single exponential. The need for a
second component was determined by a trial fit with two
components. If the fitting algorithm converged and gave a
nontrivial second component (i.e., time constant and amplitude ⬎ 0), then two components were assumed. The fitting
and number of components was determined blind to the
genotype. In the majority of cases, the number of components
was easily confirmed by visual inspection. Cell capacitance
was determined for all cells, and all currents are reported as
current densities. Cell capacitance was determined by nulling all capacitative charge in the on-cell configuration, converting to the whole cell configuration, and then immediately
determining the cell capacitance by providing the cell with a
voltage ramp and measuring the resulting current. As a
check, we integrated the amount of current passed during a
small voltage step (10 mV). The cell capacitance determined
using these two separate methods gave results within 5% of
each other.
Statistics. All comparisons were made using robust statistics (15). The robust median and the 99% confidence interval
about the robust median were determined for the parameters
of interest. The confidence interval is based on the biweight
locator and estimate of scale.
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
H1713
Fig. 1. Heart size and morphology are
not altered in slow mo (smo) mutant
adults. Histological cross sections
through adult (9 mo old) fish at the
level of the heart preserve orientation
and shape and demonstrate that slow
mo mutant hearts (B) are not grossly
different from wild-type (WT) adult
fish hearts (A). Bar, 250 ␮m.
Fig. 2. Heart rate is reduced in adult slow mo zebrafish. Adult
zebrafish were anesthetized with propofol, and the heart rate was
counted under direct vision. Heart rate counts were binned at increments of 5 beats/min (bpm), and the resulting histogram for each
genotype is shown; n ⫽ 81 WT fish (A) and 75 slow mo fish (B) for the
generation of the histograms. The 99% confidence interval is shown
by the dark gray bars in each histogram. The solid line at the center
of the bar indicates the robust median. The slow mo population had
a slower heart rate than the WT population.
AJP-Heart Circ Physiol • VOL
beats/min, 99% robust confidence interval, 32.1–41.7
beats/min). We also performed heart rate counts at a
warmer temperature (26°C) using the fish anesthetic
tricaine, which has a different molecular structure and
presumed mechanism of action from propofol. Our
earlier work (3) has shown that the heart rate is
temperature sensitive, and we wanted to explore a
temperature that was very close to the physiological
temperature at which the fish were reared. We found
that the slow mo bradycardia was not due to the effects
of a particular anesthetic or the temperature because
the bradycardia was still present when heart rates
were determined at 26°C in tricaine (wild-type: n ⫽ 28;
robust median, 111 beats/min, 99% robust confidence
interval, 106–116 beats/min; slow mo: n ⫽ 25; robust
median, 80.0 beats/min, 99% robust confidence interval, 72.2–87.9 beats/min).
In wild-type adult cardiomyocytes, ventricular Ih is
smaller than atrial Ih. In other species, it has been
noted that there are chamber-specific properties to
Ih, with a difference in current density and voltage
dependence between the atrium and ventricle that
presumably would make the ventricle less likely to
activate Ih, thereby favoring atrial pacemaking. Our
prior work using embryonic zebrafish revealed a
reduced Ih in embryonic slow mo cardiomyocytes. In
our earlier work (3), it was not technically feasible to
separately culture atrial and ventricular myocytes
from the embryonic heart, so we did not distinguish
whether chamber-specific differences were present
in the embryo. During those prior studies, we did
observe a range of current densities and activation
voltages for Ih in the mixed population of cells from
the embryonic heart, so it is possible that embryonic
cells do have chamber-specific properties. It is important to note that the differences between slow mo
and wild-type Ih are dramatic throughout the entire
voltage range of activation.
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
slightly reduced viability. As shown in Fig. 1, slow mo
mutant adults hearts are histologically indistinguishable from wild-type hearts. Overall morphology and
size are not different between wild-type fish and slow
mo mutants (data not shown).
We examined adult heart rates directly through the
skin in intact fish under anesthesia. Heart rates were
counted from a group of adult wild-type zebrafish (n ⫽
81) and compared with heart rates from an agematched group of slow mo fish (n ⫽ 75). As shown in
Fig. 2, slow mo heart rates were significantly lower
than those in wild-type hearts (wild-type: robust median, 50.5 beats/min; 99% robust confidence interval,
45.2–55.8 beats/min; slow mo: robust median, 36.9
H1714
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
Chamber-specific cell culture is feasible from the
adult fish heart (see MATERIALS AND METHODS). To investigate adult Ih, we performed whole cell patch-clamp
recordings, separately evaluating cells isolated from
the atrium or ventricle of wild-type fish hearts. Ih from
adult wild-type atrial cells is evident as a hyperpolarization-activated inward current, as shown in Fig. 3A.
A typical recording from an adult wild-type ventricular
cardiomyocyte also revealed the presence of a hyperpo-
larization-activated inward current, Ih (Fig. 3A). However, Ih in the ventricle was smaller than in the atrium,
as shown in Fig. 3A. The current density of Ih was 33.3
pA/pF in atrial cells compared with 7.6 pA/pF in ventricular cells (Table 1). As expected for Ih, bath application of CsCl (2 mM) eliminated nearly all of the
inward Ih in six wild-type atrial cells. The residual
current density was 0.3 pA/pF and amounted to 1% of
the unblocked current density.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Fig. 3. Pacemaker current (Ih) is reduced in both the atrium and ventricle of adult slow mo zebrafish. A:
representative Ih recorded from individual atrial and ventricular cells demonstrate two findings. First, slow
mo Ih are reduced in size compared with WT Ih in both the atrium and ventricle. Second, ventricular currents
are smaller than atrial currents for both WT and slow mo. Currents were elicited by hyperpolarizing the cells
from a holding voltage of ⫺40 mV to more negative voltages (⫺130 to ⫺50 mV in 10-mV increments). After the
8-s hyperpolarization, the voltage was stepped to ⫺130 mV for 1 s and then returned to ⫺40 mV. Currents
were elicited every 15 s. The data are shown without any leak subtraction, and the calibration bars pertain to
each current family. B: histograms and 99% robust confidence intervals for the current density of Ih show that
the population of slow mo cells (b) from atria (n ⫽ 39) and ventricles (n ⫽ 36) have less current density than
the population of WT cells (a) from atria (n ⫽ 36) and ventricles (n ⫽ 30). Current densities of Ih were
determined at ⫺130 mV as previously described (3). For atrial cells, current densities were binned every 5
pA/pF. The 99% robust confidence interval is shown by the dark gray bars in each histogram. The solid line
at the center of the bar indicates the robust median. In atrial cells, slow mo current density of Ih was lower
than that in WT. Note the excess number of slow mo atrial cells having little or no current density. For
ventricular cells, current densities were binned every 1.5 pA/pF. In ventricular cells, the slow mo current
density of Ih was lower than that in WT. Note the excess of slow mo ventricular cells having little or no current
density. Note the difference in the y-axis between the atrial and ventricular histograms. Atrial current density
was larger than ventricular current density for both genotypes.
AJP-Heart Circ Physiol • VOL
281 • OCTOBER 2001 •
www.ajpheart.org
SLOW MO
H1715
REDUCES PACEMAKER CURRENT AND HEART RATE
Table 1. Summary of measured current characteristics from both WT and
slow mo cells for each cardiac chamber
Atrium
n
Cm, pF
WT
slow mo
79
78
Ventricle
Robust
median
18.1
16.9
99% Robust
CI
n
16.56–19.67
15.40–18.40
78
85
Robust
median
16.3
16.7
99% Robust
CI
14.75–17.86
15.61–17.79
Ih characteristics
36
39
33.3*†‡
9.7*†‡
36
28
⫺106.8
⫺105.7
36
28
12.4
10.2
35
24
669
778
36
30
5,033
4,971
25.2–41.4
4.8–14.5
30
36
7.6*†‡
2.0*†‡
⫺110.2 to ⫺103.4
⫺110.1 to ⫺101.2
29
26
⫺105.2
⫺107
11.34–13.42
8.40–11.94
29
26
10.6
10.4
4.90–10.21
1.14–2.87
⫺110.8 to ⫺99.6
⫺112.0 to ⫺101.9
9.57–11.71
8.11–12.77
584–753
631–925
ND
ND
ND
ND
4,024–6,041
3,839–6,104
ND
ND
ND
ND
36
39
12.1*
1.1*
8.66–15.55
0.19–1.99
ND
ND
ND
ND
36
39
12.7*
5.0*
10.52–14.93
2.90–7.05
ND
ND
ND
ND
INa characteristics
Current density, pA/pF
WT
slow mo
V1/2, mV
WT
slow mo
Vs, mV
WT
slow mo
43
36
97.9
136.2†
74.5–121.2
95.1–177.3
44
46
99.3
83.5†
43
36
⫺77.6†
⫺79.8†
⫺79.4 to ⫺75.8
⫺81.9 to ⫺77.7
41
42
⫺71.1†
⫺70.3†
5.02–5.58
5.03–5.62
41
42
43
36
5.30†
5.33†
4.69†
4.61†
88.4–110.2
73.0–93.9
⫺73.0 to ⫺69.2
⫺71.5 to ⫺69.1
4.54–4.85
4.49–4.74
n ⫽ No. of cells. WT, wild-type; CI, confidence interval; Cm, cell capacitance; Ih, pacemaker current; V1/2, voltage at which current is
half-activated; Vs, voltage sensitivity of activation for current; ␶fast and ␶slow, time constants for the fast and slow components of Ih activation
at ⫺130 mV, respectively; Ampfast and Ampslow, current densities (amplitudes) for the fast and slow components of Ih activation at ⫺130 mV,
respectively; INa, sodium current. Each parameter was measured for both genotypes and both chambers. Ventricular Ih from slow mo cells
were so small that accurate kinetics could not be obtained. A meaningful comparison could, therefore, not be performed between WT and slow
mo Ih from the ventricle. ND, not determined. * Significantly different between WT and slow mo; † significantly different between atrium and
ventricle; ‡ significantly different between both WT and slow mo and atrium and ventricle.
Ih is reduced in adult slow mo atrial cardiomyocytes.
As shown in Fig. 3A, Ih in adult slow mo atrial cells is
smaller than in wild-type atrial cells. To quantitate the
difference in Ih between wild-type and slow mo cells,
we compared the current density between a group of
wild-type cells (n ⫽ 36) and a group of slow mo cells
(n ⫽ 39). The current density was significantly smaller
in slow mo atrial cells (wild-type: robust median, 33.3
pA/pF; 99% robust confidence interval, 25.2–41.4 pA/
pF; slow mo: robust median, 9.7 pA/pF; 99% robust
confidence interval, 4.8–14.5 pA/pF). A histogram of
our data along with the 99% confidence interval on the
robust median is presented in Fig. 3B. The histograms
demonstrate that the slow mo atrial cells have a lower
current density of Ih and that many slow mo cells have
no discernable Ih.
AJP-Heart Circ Physiol • VOL
We previously reported that Ih has two components:
a fast large amplitude and a slow smaller amplitude
component (3). We therefore quantitatively compared
the amplitude (Ampfast) and time constant for the fast
component (␶fast) of Ih and the amplitude (Ampslow) and
time constant for the slow component (␶slow) of Ih. We
also compared the voltage at which Ih (or INa) is half
activated (V1/2) and the voltage sensitivity of activation
(Vs). The results are summarized in Table 1. We found
that of all the parameters we examined, only the Ih
amplitudes were different between wild-type and slow
mo cardiomyocytes. Ampfast and Ampslow were reduced
in the slow mo cells compared with wild-type cells
(Ampfast of wild-type: robust median, 12.1 pA/pF; 99%
robust confidence interval, 8.7–15.6 pA/pF; Ampfast of
slow mo: robust median, 1.1 pA/pF; 99% robust confi-
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Current density, pA/pF
WT
slow mo
V1/2, mV
WT
slow mo
Vs, mV
WT
slow mo
␶fast, ms
WT
slow mo
␶slow, ms
WT
slow mo
Ampfast, pA/pF
WT
slow mo
Ampslow, pA/pF
WT
slow mo
H1716
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
AJP-Heart Circ Physiol • VOL
DISCUSSION
This study provides genetic evidence that Ih is an
essential component of cardiac pacemaking in the
adult zebrafish. A defect in the slow mo gene causes a
reduced heart rate in adult zebrafish and, at the cellular level, a reduction in the activity of Ih in both
chambers of the heart.
The electrophysiological phenotype of slow mo differs slightly between the adult and embryonic heart.
We (3) previously reported that the slow mo mutation
affects embryonic heart rate, providing evidence that
Ih is essential for pacing in the embryo. Our study of
cardiomyocytes derived from embryonic hearts revealed that the slow mo mutation appears to perturb
predominantly the fast component of Ih, leaving the
slow component unaltered (3). In the adult zebrafish
atria, however, where both the fast and slow components could be distinguished, slow mo reduces the
amplitude of both components of Ih. The time constants
for each component are unchanged, but the amplitude
of the currents contributed by the fast as well as the
slow components are reduced (Table 1). A comparison
of the kinetics of embryonic wild-type Ih with adult
atrial Ih shows a slowing of both fast and slow components in the adult compared with the embryo. An
alteration in our recording solutions and the inclusion
of tetraethylammonium in the bath solution for recording the adult Ih may have played at least some part in
slowing adult Ih kinetics. A more likely contributing
factor is the fact that the voltage range over which the
adult cells activate is nearly 13 mV more negative than
that in embryonic cells. This would tend to slow the
adult kinetics for any given voltage step. A similar
slowing has been noted during development of rat
ventricular Ih (27). Despite the reduced amplitude of
slow mo Ih, Ih still responds to internal cAMP by
shifting the voltage at which it activates into a more
depolarized region. In this regard, slow mo Ih behaves
like the wild-type Ih in both the atrium and ventricle.
Genes encoding Ih channels have recently been
cloned. These genes were termed hyperpolarizationactivated cyclic nucleotide-gated (HCN) genes for the
properties of the channels they encode (8, 13, 21, 28).
HCN channels are members of the voltage-gated K⫹
channel family and possess a COOH-terminal cyclic
nucleotide-binding domain. Expression studies of HCN
family members have revealed that HCN isoforms
have different activation kinetics, suggesting that the
different kinetic components of Ih measured in cardiomyocytes reflect individual contributions from separate HCN isoforms (22). Perhaps the slow mo mutation
affects gene products in a developmentally differential
pattern and the embryonic Ih profile is differentially
affected compared with the adult. A change of isoform
profiles in development has been documented for Ih in
the rat ventricle (29).
We found that atrial cardiomyocytes in the zebrafish
heart have a greater current density of Ih than ventricular cardiomyocytes, as is true for other vertebrates.
This has been teleologically ascribed to the beneficial
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
dence interval, 0.2–2.0 pA/pF; Ampslow of wild-type:
robust median, 12.7 pA/pF; 99% robust confidence interval, 10.5–14.9 pA/pF; Ampslow of slow mo: robust
median, 5.0 pA/pF; 99% robust confidence interval,
2.9–7.1 pA/pF). In contrast, we found no significant
difference between V1/2, Vs, ␶fast, or ␶slow for Ih (Table 1).
We also tested both wild-type and slow mo atrial cardiomyocytes for their responsiveness to internal cAMP.
We included 100 ␮M cAMP in the patch pipette and
found that V1/2 of Ih markedly shifted in the depolarized direction (wild-type: n ⫽ 48; robust median, ⫺84.0
mV; 99% robust confidence interval, ⫺85.9 to ⫺82.0
mV; slow mo: n ⫽ 39; robust median, ⫺87.7 mV; 99%
robust confidence interval, ⫺90.2 to ⫺85.2 mV).
Ih is reduced in adult slow mo ventricular cardiomyocytes. Even though wild-type ventricular Ih is small
compared with atrial Ih, we could still observe a diminution of ventricular Ih in the slow mo mutant (Fig.
3A). The current density of Ih was smaller in slow mo
ventricular cells compared with wild-type cells (wildtype: n ⫽ 30; robust median, 7.6 pA/pF; 99% robust
confidence interval, 4.9–10.2 pA/pF; slow mo: n ⫽ 36;
robust median, 2.0 pA/pF; 99% robust confidence interval, 1.1–2.9 pA/pF). A histogram of these data along
with the 99% confidence interval on the robust median
is shown in Fig. 3B. The histograms demonstrate that
slow mo ventricular cells have a lower current density
of Ih and that many slow mo ventricular cells have no
discernable Ih. Because of the small size of ventricular
Ih, we were not able to confidently perform a kinetic
analysis of Ih activation, but, as shown in Table 1, we
found no significant difference between wild-type and
slow mo Ih in terms of V1/2 or Vs (Table 1). We also
tested both wild-type and slow mo ventricular cardiomyocytes for their responsiveness to internal cAMP.
We included 100 ␮M cAMP in the patch pipette and
found that V1/2 markedly shifted in the depolarized
direction (wild-type: n ⫽ 8; robust median, ⫺75.3 mV;
99% robust confidence interval, ⫺88.1 to ⫺62.5 mV;
slow mo: n ⫽ 6; robust median, ⫺80.5 mV; 95% robust
confidence interval, ⫺92.7 to ⫺68.2 mV).
The slow mo mutation has minimal effects on INa in
adult zebrafish. As a control for the specificity of the
effects of the slow mo mutation on ion currents other
than Ih, we examined the inward INa, found in nearly
all of the cells of both slow mo and wild-type hearts.
Typical recordings from these cardiomyocytes are
shown in Fig. 4A. In our wild-type chamber-specific
analysis, we found some distinctions between ventricular and atrial INa in zebrafish hearts. Atrial INa has a
steady-state inactivation voltage that is more negative
than ventricular INa, and there is a reduced voltage
sensitivity to steady-state inactivation with atrial INa
compared with ventricular INa (Table 1). However,
these features were not different between wild-type
and slow mo mutant cells. In addition, we found no
significant differences in the current density at ⫺30
mV of atrial INa between wild-type (n ⫽ 43) and slow
mo cells (n ⫽ 36) or of ventricular INa between wildtype (n ⫽ 44) and slow mo cells (n ⫽ 46) (Table 1 and
Fig. 4B).
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
H1717
effect of restricting pacemaking to pacemaking tissues,
thereby avoiding ectopic pacemaking. The specific
strategy for Ih distribution patterning appears to differ
from species to species. Human, mouse, rabbit, and rat
cardiomyocytes manifest a negative shift of the voltage
dependence of activation of Ih in tissues where pacemaking is usually inappropriate (12, 27, 29). This is not
true for zebrafish cardiomyocytes, in which the atrial
and ventricular cells differ not in the activation voltage
for Ih but rather in the current density over the entire
activation range. This has also been reported in the
rat, in which ventricular current density of Ih is reAJP-Heart Circ Physiol • VOL
duced compared with atrial current density, without a
shift in activation voltage (6).
How the slow mo gene normally affects Ih must await
its cloning. Although it is formally possible that slow
mo is an Ih gene, our preliminary linkage studies do not
suggest this to be the case. Alternatively, it might be a
modifier of the function of the channel. Most known
modifiers of Ih increase or decrease Ih activity via a
shift in the voltage dependence of activation. This is
true, for example, of adrenergic and cholinergic agonists (11), calcium (23), substance P via the NK1 receptor (17), and opioids (16, 33). Modifications of native Ih
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
Fig. 4. Sodium current (INa) is not affected in either the atrium or ventricle by the slow mo mutation. A:
representative INa recorded from individual atrial and ventricular cells demonstrate two findings. First, slow mo
INa are the same size compared with WT in both the atrium and ventricle. Second, the ventricular currents have
slower kinetics than atrial currents for both WT and slow mo. All currents were elicited by stepping the voltage to
⫺30 mV after a 1-s prepulse to voltages ranging from ⫺120 to ⫺10 mV in 10-mV increments. The prepulse results
in steady-state inactivation, which is then measured by a test pulse to ⫺30 mV. Currents were elicited every 3 s.
The data are shown without any leak subtraction, and the calibration bars pertain to each current family. B:
histograms and 99% robust confidence intervals for current density of INa are shown for each chamber and
genotype. a: WT cells; b: slow mo cells. INa was determined by plotting the peak inward current at ⫺30 mV as a
function of prepulse voltage and fitting the resulting curve with a Boltzmann function. The current density was
then obtained by dividing the current amplitude at ⫺30 mV (as determined from the fit) by the cell capacitance.
Because the current amplitude was determined at a fixed voltage (⫺30 mV), leak current and nonlinear
current-voltage issues were avoided. For all cells, current densities were binned every 25 pA/pF. The 99% robust
confidence interval is shown by the dark gray bars in each histogram. The solid line at the center of the bar
indicates the robust median. In both atrial and ventricular cells, slow mo current density of INa was not statistically
different than that in WT.
H1718
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
that alter the size of Ih without changing the activation
voltage or kinetics, as is true of the slow mo mutation,
include inhibition of phosphatase activity (1) and the
activation of epidermal growth factor receptor tyrosine
kinase (38). Perhaps the slow mo mutation affects the
phosphorylation state of an HCN channel isoform or
the phosphorylation state of a modifier of the Ih channel. The phosphorylation state of modifier proteins has
recently been shown to affect channel activity of the
skeletal ryanodine (RYR) channel [calsequestrin (34)]
and the cardiac RYR2 receptor [FKBP12.6 (25)].
REFERENCES
1. Accili EA, Redaelli G, and DiFrancesco D. Differential control of the hyperpolarization-activated curent [i(f)] by cAMP
gating and phosphatase inhibition in rabbit sino-atrial node
myocytes. J Physiol 500: 643–651, 1997.
2. Alexander J, Stainier DY, and Yelon D. Screening mosaic F1
females for mutations affecting zebrafish heart induction and
patterning. Dev Genet 22: 288–299, 1998.
3. Baker K, Warren KS, Yellen G, and Fishman MC. Defective
“pacemaker” current (Ih) in a zebrafish mutant with a slow heart
rate. Proc Natl Acad Sci USA 94: 4554–4559, 1997.
4. Burggren W and Doyle M. Ontogeny of heart rate regulation
in the bullfrog, Rana catesbeiana. Am J Physiol Regulatory
Integrative Comp Physiol 251: R231–R239, 1986.
5. Burggren WW and Pinder AW. Ontogeny of cardiovascular
and respiratory physiology in lower vertebrates. Annu Rev
Physiol 53: 107–135, 1991.
6. Cerbai E, Pino R, Sartiani L, and Mugelli A. Influence of
postnatal development on If occurrence and properties in
neonatal rat ventricular myocytes. Cardiovasc Res 42: 416–
423, 1999.
7. Chen JN, Haffter P, Odenthal J, Vogelsang E, Brand M,
van Eeden FJM, Furutani-Seiki M, Granato M, Hammerschmidt M, Heisenberg CP, Jiang YJ, Kane DA, Kelsh RN,
Mullins MC, and Nüsslein-Volhard C. Mutations affecting
the cardiovascular system and other internal organs in zebrafish. Development 123: 293–302, 1996.
8. Clapham DE. Not so funny anymore: pacing channels are
cloned. Neuron 21: 5–7, 1998.
9. DiFrancesco D, Ducouret P, and Robinson RB. Muscarinic
modulation of cardiac rate at low acetylcholine concentrations.
Science 243: 669–671, 1989.
10. DiFrancesco D, Ferroni A, Mazzanti M, and Tromba C.
Properties of the hyperpolarizing-activated current (if) in cells
isolated from the rabbit sino-atrial node. J Physiol (Lond) 377:
61–88, 1986.
11. DiFrancesco D, Mangoni M, and Maccaferri G. The pacemaker current in cardiac cells. In: Cardiac Electrophysiology,
from Cell to Bedside, edited by Zipes DP and Jalife J. Saunders, Philadelphia, PA: 1995.
12. Fares N, Bois P, Lenfant J, and Potreau D. Characterization
of a hyperpolarization-activated current in dedifferentiated
adult rat ventricular cells in primary culture. J Physiol (Lond)
506: 73–82, 1998.
13. Gauss R, Seifert R, and Kaupp UB. Molecular identification
of a hyperpolarization-activated channel in sea urchin sperm.
Nature 393: 583–587, 1998.
AJP-Heart Circ Physiol • VOL
281 • OCTOBER 2001 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
We thank Chris Simpson for excellent histological assistance.
This work was supported by National Institutes of Health Grants
R01-HL-49579, R01-DK-55383, and R01-HL-63206 (to M. C. Fishman), by a New Investigator Award from the Foundation for Anesthesia Education and Research and the Society for Cardiovascular
Anesthesiologists (to K. Baker), and by The Howard B. Sprague
Fellowship, American Heart Association, Massachusetts Affiliate (to
K. S. Warren).
Present address of K. S. Warren: Dept. of Biology and Marine
Biology, Roger Williams Univ., Bristol, RI 02809.
14. Gerhart J and Kirschner M. Cells, Embryos, and Evolution.
Malden, MA: Blackwell Science, 1997.
15. Iglewicz B. Robust scale estimators and confidence intervals for
location. In: Understanding Robust and Exploratory Data Analysis, edited by Hoaglin DC, Mosteller F, and Tukey JW. New
York: Wiley, 1983, p. 404–429.
16. Ingram SL and Williams JT. Opioid inhibition of Ih via adenylyl cyclase. Neuron 13: 179–186, 1994.
17. Jafri MS and Weinreich D. Substance P regulates Ih via a
NK-1 receptor in vagal sensory neurons of the ferret. J Neurophysiol 79: 769–777, 1998.
18. Keller BB. Overview: functional maturation and coupling of the
embryonic cardiovascular system. In: Developmental Mechanisms of Heart Disease. Armonk, NY: Futura, 1995, p. 367–385.
19. Lillywhite HB, Zippel KC, and Farrell AP. Resting and
maximal heart rates in ectothermic vertebrates. Comp Biochem
Physiol A Mol Integr Physiol 124: 369–382, 1999.
20. Liu ZW, Zou AR, Demir SS, Clark JW, and Nathan RD.
Characterization of hyperpolarization-activated inward current
in cultured pacemaker cells from the sinoatrial node. J Mol Cell
Cardiol 21: 2523–2535, 1996.
21. Ludwig A, Zong X, Jeglitsch M, Hofmann F, and Biel M. A
family of hyperpolarization-activated mammalian cation channels. Nature 393: 587–591, 1998.
22. Ludwig A, Zong X, Stieber J, Hullin R, Hofmann F, and
Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J 18: 2323–2329,
1999.
23. Luthi A and McCormick DA. H-current: properties of a neuronal and network pacemaker. Neuron 21: 9–12, 1998.
24. Maruoka F, Nakashima Y, Takano M, Ono K, and Noma A.
Cation-dependent gating of the hyperpolarization-activated cation current in the rabbit sino-atrial node cells. J Physiol (Lond)
477: 423–435, 1994.
25. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff
D, Rosembilt N, and Marks AR. PKA Phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine
receptor): defective regulation in failing hearts. Cell 101: 365–
376, 2000.
26. Mitra R and Morad M. A uniform enzymatic method for dissassociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol Heart Circ Physiol 249: H1056–H1060,
1985.
27. Robinson RB, Yu H, Chang F, and Cohen IS. Developmental
change in the voltage-dependence of the pacemaker current, if, in
rat ventricle cells. Pflugers Arch 433: 533–535, 1997.
28. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER,
Siegelbaum SA, and Tibbs GR. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain.
Cell 93: 717–729, 1998.
29. Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D, and Cohen IS. Distribution
and prevalence of hyperpolarization-activated cation channel
(HCN) mRNA expression in cardiac tissues. Circ Res 85: e1–e6,
1999.
30. Shigenobu K. Electrophysiological properties of chick embryonic heart and some pharmacological studies with rat myocardium during pre- and postnatal development. In: Developmental
Cardiology: Morphogenesis and Function, edited by Clark EB
and Takao A. Mount Kisco, NY: Futura, 1990, p. 273–289.
31. Solomon JS and Nerbonne JM. Two kinetically distinct components of hyperpolarization-activated current in rat superior
colliculus-projecting neurons. J Physiol (Lond) 469: 291–313,
1993.
32. Stainier DYR, Fouquet B, Chen JN, Warren K, Weinstein
B, Meiler S, Mohideen MAPK, Neuhauss SCF, SolnicaKrezel L, Schier AF, Zwartkruis F, Stemple DL, Malicki J,
Driever W, and Fishman MC. Mutations affecting the formation and function of the cardiovascular system in zebrafish
embryos. Development 123: 285–292, 1996.
33. Svoboda KR and Lupica CR. Opioid inhibition of hippocampal
interneurons via modulation of potassium and hyperpolarization-activated cation (Ih) currents. J Neurosci 18: 7084–7098,
1998.
SLOW MO
REDUCES PACEMAKER CURRENT AND HEART RATE
34. Szegedi C, Sarkozi S, Herzog A, Jona I, and Varsanyi M.
Calsequestrin: more than “only” a luminal Ca2⫹ buffer inside the
sarcoplasmic reticulum. Biochem J 337: 19–22, 1999.
35. Warren KS and Fishman MC. “Physiological genomics”: mutant screens in zebrafish. Am J Physiol Heart Circ Physiol 275:
H1–H7, 1998.
36. Warren KS, Wu JC, Pinet F, and Fishman MC. The genetic basis of cardiac function: dissection by zebrafish (Danio
H1719
rerio) screens. Philos Trans R Soc Lond B Biol Sci 355:
939–944, 2000.
37. Westerfield M. The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish Danio rerio. Eugene, OR: University of
Oregon, 1995.
38. Wu J, Yu H, and Cohen IS. Epidermal growth factor increases
i(f) in rabbit SA node cells by activating a tyrosine kinase.
Biochim Biophys Acta 1463: 15–19, 2000.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on June 17, 2017
AJP-Heart Circ Physiol • VOL
281 • OCTOBER 2001 •
www.ajpheart.org