doi: 10.1161/hh1301.094395
© 2001 American Heart Association, Inc.
UltraRapid Communication |
HCN2 Overexpression in Newborn and Adult Ventricular Myocytes
Distinct Effects on Gating and Excitability
From the Department of Pharmacology (J.Q., A.B., L.P., R.B.R.), Center for Molecular Therapeutics (I.S.C., R.B.R.) and Center for Neurobiology and Behavior (B.S.), Columbia University, New York, and Department of Physiology and Biophysics and Institute of Molecular Cardiology (I.S.C.), State University of New York, Stony Brook.
Correspondence to Richard B. Robinson, PhD, Department of Pharmacology, Columbia University, 630 W 168th St, New York, NY 10032. E-mail rbr1{at}columbia.edu
Abstract
Abstract—Ventricular pacemaker current (If) shows distinct voltage dependence as a function of age, activating outside the physiological range in normal adult ventricle, but less negatively in neonatal ventricle. However, heterologously expressed HCN2 and HCN4, the putative molecular correlates of ventricular If, exhibit only a modest difference in activation voltage. We therefore prepared an adenoviral construct (AdHCN2) of HCN2, the dominant ventricular isoform at either age, and used it to infect neonatal and adult rat ventricular myocytes to investigate the role of maturation on current gating. The expressed current exhibited an 18-mV difference in activation (V1/2 -95.9±1.9 in adult; -77.6±1.6 mV in neonate), comparable to the 22-mV difference between native If in adult and neonatal cultures (V1/2 -98.7 versus -77.0 mV). This did not result from developmental differences in basal cAMP, because saturating cAMP in the pipette caused an equivalent positive shift in both preparations. In the neonate, AdHCN2 caused a significant increase in spontaneous rate compared with control (88±5 versus 48±4 bpm). In adult, where HCN2 activates more negatively, the effect was evident only during anodal excitation, requiring significantly less stimulus energy than control (2149±266 versus 3140±279 mV · ms). Thus, ventricular maturational state influences the voltage dependence of expressed HCN2, resulting in distinct physiological impact of expressed channels in neonate and adult myocytes. The full text of this article is available at http://www.circresaha.org.
Key Words: pacemaker current • gene expression • development • ventricle • HCN
The recent cloning of the HCN gene family, representing the molecular correlate of the pacemaker current If, raises the possibility of using genetic approaches for the treatment of rhythm disorders. However, If is present in both automatic1 and nonautomatic2 3 4 5 6 regions of the heart, and the threshold voltage varies widely among cardiac regions, being least negative in sinus node (-40 mV in rabbit7 ) and most negative in ventricle (-108 mV or more negative5 8 9 ). Interestingly, If activates at less negative voltages in newborn ventricle (-70 mV in rat8 10 ). The molecular and cellular bases for the regional variability of activation voltages in the normal adult heart and the regulation of ventricular activation voltage by development remain to be determined, but such understanding is critical to any future therapeutic application of the expressed current in myocardium.
Four members of the HCN gene family are currently known.11 12 13 Three of these (HCN1, HCN2, and HCN4) are present in heart, and their relative message level varies with region and age.14 15 Sinus node and Purkinje fibers, in which If activates at less negative potentials, contain largely HCN1 and HCN4. Ventricle contains HCN2 and HCN4, with the HCN2/HCN4 mRNA ratio being greater in adult than newborn. This suggests that HCN2 is inherently negatively activating, and its relative abundance determines the activation threshold in different regions of the heart or at different ages. However, when HCN2 and HCN4 were expressed in mammalian cell lines, activation voltages differed by <10 mV.16 17 Thus, the intrinsic voltage dependence of different HCN isoforms is insufficient to explain the functional diversity of native cardiac If, either regionally or developmentally. Instead, it seems likely that unidentified accessory proteins or other cellular factors influence the voltage dependence of individual HCN isoforms. Thus, HCN2 activation might differ, for example, when expressed in neonatal versus adult ventricular myocytes. The present study investigated this possibility.
Materials and Methods
Cell Isolation and Culture
Adult rats were anesthetized with
ketamine-xylazine before cardiectomy, and neonatal rats
decapitated, in accordance with Institutional Animal Care and Use
Committee protocols of Columbia University. A standard trypsin
dissociation method was used to prepare newborn ventricle cell
cultures.18 The cells were
preplated to reduce fibroblast proliferation, cultured initially in
serum-containing medium, then switched to serum-free medium after 24
hours. Action potential studies were conducted on 4- to 6-day-old
monolayer cultures plated directly onto fibronectin-coated 9x22-mm
glass coverslips. For voltage-clamp experiments, 4- to 6-day-old
monolayer cultures were resuspended by brief (2 to 3 minutes) exposure
to 0.25% trypsin, then the cells replated onto fibronectin-coated
coverslips and studied within 2 to 8 hours.
Freshly isolated adult ventricle myocytes were prepared using the procedure described by Kuznetsov et al.19 This entailed a Langendorff perfusion of collagenase, followed by trimming away of the atria. The remaining tissue was minced and dissociated in additional collagenase solution. The isolated myocytes were suspended in a serum-free medium (ACCTI20 ) then plated onto laminin-coated 9x22-mm glass coverslips at 0.5 to 1x103 cells/mm2. Two to three hours later, after the myocytes had adhered to the coverslips, the adenoviral infection procedure was begun.
Expression of HCN2
For increased expression efficiency, we prepared an
adenoviral construct of mouse HCN2 (mHCN2,
GenBank No. AJ225122) following previously
published methods.21 A DNA
fragment (between EcoRI and
XbaI restriction sites) that
included mHCN2 DNA downstream of the cytomegalovirus promoter was
obtained from plasmid
pTR-mHCN222 and subcloned
into the shuttle vector pDC516 (AdMax; Microbix
Biosystems). The resulting pDC516-mHCN2 shuttle plasmid was
cotransfected with a 35.5-kb E1-deleted adenoviral genomic plasmid
pBHGfrt
E1,3FLP (AdMax) into E1-complementing
HEK293 cells. The adenoviral construct AdHCN2 was subsequently
harvested and purified with CsCl.
AdHCN2 infection of adult rat ventricular myocytes was carried out 2 to 3 hours after cell plating. Infection of neonatal cells was done on monolayer cultures 1 to 3 days after plating. In either case, the culture medium was removed from the 35-mm dishes and the inoculum of 0.2 to 0.3 mL/dish was added containing AdHCN2. The multiplicity of infection (MOI; ratio of viral units to cells) was 15 to 100. The inoculum was dispersed over the cells by gently tilting the dishes every 20 minutes. The dishes were kept at 37°C in a CO2 incubator during the 2-hour adsorption period, then the inoculum was discarded and the dishes washed and refilled with the appropriate culture medium. The dishes remained in the incubator for 24 to 48 hours before electrophysiological experiments were conducted. In some experiments, a green fluorescent protein (GFP) adenoviral construct (AdGFP) was used as control.
Electrophysiological
Recording and Data Analysis
Native
If,
expressed
IHCN2,
and action potentials were recorded using a patch electrode in
whole-cell mode on cells superfused at 35°C. Extracellular solution
contained (in mmol/L) NaCl 140, NaOH 2.3,
MgCl2 1, KCl 5.4, CaCl2
1.0, HEPES 5, and glucose 10 (pH 7.4). To record
If or
IHCN2,
[K+]o was increased
to 10 mmol/L, and MnCl2 (2 mmol/L) and
BaCl2 (4 mmol/L) added to the
superfusate to eliminate calcium and inward rectifier
(IK1)
currents. In some experiments, CsCl (4 mmol/L) was used
extracellularly to define the Cs-sensitive pacemaker current. Pipette
solution included (in mmol/L) aspartic acid 130, KOH 146, NaCl 10,
CaCl2 2, EGTA-KOH 5, Mg-ATP 2, and HEPES-KOH 10
(pH 7.2). Where indicated, cAMP was included in the pipette solution. A
fast solution–changing apparatus expedited the
experimental protocols. The pipette resistance was 1 to 3 M
. An
Axopatch-200B amplifier and pClamp8 software (Axon
Instruments) were used for acquisition and
analysis.
Pacemaker current
(If or
IHCN2)
was defined as the time-dependent component at the end of a 3- to
6-second hyperpolarizing step (range -35 to -145 mV) from a holding
potential of -35 mV, unless otherwise indicated. The hyperpolarizing
step was followed by an 8-second step to -125 mV to record tail
current, then a 0.5-second pulse to -5 mV to ensure full
deactivation. Tail currents were measured at -125 mV to avoid
contamination by currents present at less negative voltages. To
minimize contamination of expressed current by native
If,
cells from infected cultures were only included if the measured current
at -125 mV was 
200 pA. This resulted in discarding <8% of cells
from AdHCN2-infected cultures at either age. In addition, all 12
noninfected adult cells and 8 of 9 noninfected neonatal cells used in
the measurement of native
If had
current values below this criterion. Tail current, plotted against test
voltage, provided maximum conductance and the activation-voltage
relation; the latter was normalized by maximum conductance and
fitted with the Boltzmann function
{y=1/[1+exp[(V-V1/2)/K]]} to determine
the half-maximum activation voltage (V1/2) and
slope factor (K). Kinetics of activation were determined by a
single-exponential fit to the early time course of the current
activated by hyperpolarization; the initial
delay and any late slow activation were ignored. Kinetics of
deactivation were determined by a single-exponential fit of the time
course of the current trace at each test voltage after activation by a
prepulse to -125 mV. For both activation and deactivation, the
duration of the trace being fit was 3x the measured time constant to
ensure accuracy. Records were not corrected for liquid junction
potential (9.8 mV).
All data are presented as mean±SEM. Statistical significance was examined by t test for paired and ANOVA for multiple comparisons. Values of P<0.05 were determined to be significant.
Results
Comparison of Neonatal and Adult
Ventricular Myocytes Expressing HCN2
Comparing the characteristics of HCN2 (the major
ventricular HCN isoform, at the message level, at both
ages14 ) when expressed in
adult versus neonatal ventricular myocytes required
maintaining adult cells in culture for 48 hours. Because a previous
report indicated that longer culture conditions resulted in a marked
positive shift in activation of native
current,9 we first compared
native
If
between acutely dissociated cells and cells maintained in serum-free
medium for 2 days. All cells studied were rod-shaped and quiescent. In
adult cells in culture,
If
threshold voltage (ie, first voltage step where a time-dependent
current is apparent) was relatively negative
(Figure 1A
) compared with that reported in
neonate.23 A 2-day culture
period resulted in no significant difference in
V1/2 (-105.3±2.6, n=12, versus -98.7±1.8
mV, n=7, in acute versus cultured cells;
P=0.092), slope factor
(10.9±1.2 versus 14.4±1.9 mV), or activation kinetics (data not
shown). Thus, short-term culture of adult cells can be used in studies
of age-dependent differences in
If or
IHCN2.
|
Treatment of both adult and neonatal cells in culture with
the AdHCN2 construct resulted in expression of high current levels,
with faster activation in neonate
(Figures 1B
and 1C; note different scales than in
Figure 1A).
Figure 2A illustrates the average activation relations in
myocytes expressing HCN2 in the two preparations. It is evident that,
when the same protein is expressed in newborn and adult myocytes, the
resultant current activates at significantly more negative
voltages in adult cells. V1/2 for
IHCN2 in
neonate and adult myocytes was -77.6±1.6 (n=24) and -95.9±1.9 mV
(n=13), respectively
(P<0.001). K also differed
significantly (9.8±0.6 versus 6.5±0.5 mV,
P<0.001), reflecting a more
shallow voltage dependence in neonate.
Figure 2B illustrates the activation/deactivation kinetics
for
IHCN2.
The data were well fit by a standard kinetic model (see legend) and
exhibit little difference in the maximal value of activation time
constant (
) with age. However, the voltage dependence of the
relation is shifted negatively in adult by 21 mV, comparable to the
shift in the activation relation (18 mV). Moreover, the relative peaks
of the kinetic relations in the two preparations are consistent
with the previously determined V1/2 values
(arrows,
Figure 2B). For both expressed
IHCN2
and native
If
(Figure 2C), kinetics in neonatal and adult myocytes appear
related to the voltage dependence of steady-state
activation.
|
Possible Basis for Difference Between Neonatal
and Adult Myocytes Expressing HCN2
The biophysical characteristics of heterologously
expressed currents can vary with current
density.24 25 26
To determine if the difference in V1/2 of HCN2
between neonatal and adult resulted from this phenomenon, we conducted
a linear regression analysis
(Figure 3). The results indicate that differences in
expression level cannot explain the difference in HCN2 voltage
dependence between neonatal and adult myocytes. Neonatal myocytes
exhibited a wide range of current density for expressed current, with a
correlation coefficient for V1/2 of 0.51
(P=0.01); current density was
less variable in adult, with no correlation with
V1/2 (correlation coefficient 0.043,
P=0.88). Considering current
densities common to both preparations (ie, <60 pA/pF,
Figure 3, inset), expressed current in neonatal myocytes
demonstrated a significantly less negative V1/2
than in adult myocytes
(P<0.001).
|
Both native
If and
expressed HCN2 respond to cAMP by a
phosphorylation-independent shift in voltage dependence
of
activation,27 28
although phosphorylation-dependent mechanisms also have
been reported.29 To test if
the neonatal/adult difference in V1/2 of
IHCN2
simply reflected differences in basal cAMP, we repeated the
determination of V1/2 of expressed current with
10 µmol/L cAMP in the pipette solution to eliminate any differences
in intracellular cAMP levels and achieve a maximal positive shift of
IHCN2
(Figure 4).
IHCN2
shifted positively by a comparable amount in both preparations and the
difference in V1/2 persisted, indicating that
the age-dependent difference in HCN2 activation does not result from a
difference in basal cAMP. Results with a 10x higher cAMP concentration
did not differ statistically (data not shown).
|
Functional Effect of Overexpression of
HCN2
AdHCN2 infection resulted in expression of a large
current in >90% of the cells studied. Given the activation of
IHCN2
within the physiological voltage range in neonatal
cells, we asked if HCN2 overexpression resulted in a change in
spontaneous rate of these cultures. Experiments were conducted on
monolayer cultures of synchronously beating cells, with a whole-cell
patch electrode recording from one cell of the contiguous
monolayer. Control (noninfected) cultures beat spontaneously, with a
mean rate of 48.4±4.4 bpm (n=17). There was little or no
diastolic depolarization between action potentials, and
cycle length tended to vary from beat to beat
(Figure 5A). Maximum diastolic potential (MDP)
was -65.2±1.8 mV (n=17). In contrast, AdHCN2-infected cultures
exhibited a more regular and faster rhythm
(Figure 5B), with a mean rate of 88.0±5.4 bpm (n=16), a
marked diastolic depolarization, and less negative MDP
(-42.5±1.6 mV, n=11;
Figure 5C). The differences in frequency, phase 4 slope, and
MDP were significant (P<0.05).
AdGFP-infected cultures did not differ from uninfected control cultures
but differed significantly from AdHCN2-infected cultures.
|
As expected, given the negative activation relation of
If and
IHCN2 in
adult, neither control nor AdHCN2-infected adult cultures beat
spontaneously. However, because Ranjan et
al30 31 have
proposed that native
If in
adult ventricle contributes to anode break stimulation, we hypothesized
that adult cells overexpressing HCN2 would be more susceptible to this
phenomenon. We measured the most negative potential achieved for a
threshold 20-ms anodal stimulation and the
If
density at the end of a 2-second voltage step to -125 mV in the same
cells. As predicted, infected cells more readily exhibited anode break
excitation.
Figure 6A illustrates representative traces
of anodal stimuli and resulting action potential upstrokes from control
and infected cells. The delay between the end of the anodal stimulus
and action potential threshold was not statistically different between
preparations (45±10 versus 58±9 ms,
P>0.05).
Figure 6B graphs the relation between maximal negative
potential at threshold and
If or
IHCN2
density for control and infected cells. Control cells exhibited an
inverse correlation
(Figure 6B, inset), consistent with the hypothesis
that native
If
contributes to anode break excitation. In comparison, in infected cells
it was sufficient to hyperpolarize the membrane to 
-80 mV, ie, the
threshold for expressed current. Anode break threshold was independent
of IHCN2
density, indicating the current was large enough in all infected cells
to generate a sufficient overshoot for achieving excitation at
IHCN2
threshold. Required stimulus energy (the integral of the area from
stimulus start to action potential threshold) was significantly less in
AdHCN2-infected cells (2149±266, n=12 versus 3140±279 mV · ms,
n=10; P<0.05). The required
stimulus energy of AdGFP-infected cells did not differ from control
(data not shown). In addition, resting potential did not differ between
control, AdHCN2-infected, and AdGFP-infected myocytes (data not
shown).
|
Discussion
This study investigated if the distinct activation voltage of If in neonatal and adult ventricle was the result of a difference in the biophysical properties of the HCN2 isoform when expressed in neonatal and adult ventricular myocytes. At both ages, HCN2 is the dominant isoform based on RNase protection, although the relative ratio of HCN2/HCN4 message increases developmentally.14 The results indicate that HCN2 expression in neonatal and adult ventricular myocytes in culture results in a comparable difference (18 mV) in V1/2 to that of native If (22 mV). Thus, the developmental difference in pacemaker current voltage dependence under our experimental conditions is largely accounted for by an effect of the myocyte maturational state on the HCN2 isoform. Further, this difference in activation voltage results in a marked difference in the physiological impact of expressed IHCN2 because of the relative position of the current threshold with respect to MDP as a function of age.
HCN2 in neonatal ventricle activates at less negative voltages than in other mammalian systems, with a V1/2 of -78 mV in the present study, compared with values of -83 to -99 mV.16 17 32 Although this might suggest that neonatal myocytes provide a unique environment, relative to alternative expression systems, at least one oocyte expression study33 reported HCN2 activation equivalent to that in neonatal ventricle, suggesting other systems also are capable of expressing HCN2 with less negative voltage dependence. The actual V1/2 of native If in ventricle was -77 and -99 mV in neonate and adult, respectively, compared with values for HCN2 of -78 and -96 mV in these two preparations. Thus, HCN2 activation largely explains the voltage dependence of native ventricular If. The current referred to in this study as IHCN2 is of course a sum of expressed and native current. However, selection criteria were established (see Materials and Methods) to minimize contamination by native current. Further, if native and expressed current differ significantly, then one would predict a marked dependence of activation voltage on current density, whereas the observed correlation was modest and did not account for the differences in newborn and adult.
The kinetic characteristics of the native current in neonate
and adult ventricle also are largely, but not necessarily entirely,
explainable by HCN2. Native
If in
the neonate activates with kinetics close to that of
IHCN2 in
these same cells. When the full activation/deactivation relation of
expressed HCN2 is compared in neonate and adult, the difference is
largely attributable to the difference in voltage dependence of
activation. Thus, there does not appear to be an effect of maturational
state of the myocyte directly on activation kinetics of expressed
current, independent of the effect on voltage dependence of activation.
However, native
If
kinetics appear slower than expressed HCN2 kinetics, especially in
adult (compare
Figures 2B and 2C), and this may reflect the influence of the
more slowly activating HCN4 isoform. Whether HCN2 and HCN4 can form
heteromultimers is not known, but recent studies of HCN1 and
HCN2 support the idea of heteromeric
formation.33 34
The basis for the age-dependent difference in HCN2 voltage dependence in myocytes is unclear, beyond the observation that it is not secondary to differences in basal cAMP. The range of voltage dependence reported for If in different cardiac regions or as a function of age is pronounced and may reflect a combination of mechanisms. Studies of other channels have identified several factors that alter the biophysical properties of native or expressed current, including ß subunits,35 36 local membrane composition,37 cytoskeletal interactions,38 phosphorylation/dephosphorylation,29 39 and truncation.40 A ß subunit for the HCN gene family has recently been identified,41 but it is concentrated in the sinoatrial node of the heart and affects kinetics rather than voltage dependence. The extent to which other ß subunits or the other potential mechanisms contribute to the variation in voltage dependence of If or IHCN2 is unknown. In this context, it is interesting that when native If is studied in cell-free macropatches, activation shifts markedly negatively, but treatment of the intracellular face of the patch with pronase shifts activation positively again by 56 mV.42 Further, when a large portion of the HCN2 C-terminal that includes the cyclic nucleotide-binding domain is deleted, activation shifts positively by 24 mV.43 From these results, one can speculate that interactions between cytoplasmic elements of the HCN protein contribute to more negative activation, but that these interactions are minimized in the intact cell. Although the relevance of these observations to the regional or developmental variation in activation voltage remains to be determined, it is likely that any cellular factor(s) contributing to the less negative activation of HCN2 in the neonate are not substrate limited because we observed no negative shift in V1/2 (and in fact a slight positive trend) at expression levels that were 2 to 3 orders of magnitude greater than that typical of native current.
Under our experimental conditions,
V1/2 of native current in newborn and adult
ventricle differed by 22 mV, which is lower than the previously
reported difference in threshold value of 40
mV8 In part, this may result
from the 48-hour culture period, because acutely isolated adult
myocytes had a V1/2 that was more negative by 6
mV. Although not a statistically significant difference, it is
consistent with an earlier report that extended culture of
adult myocytes under conditions that cause morphological
dedifferentiation results in a marked positive shift of activation
voltage.9 In addition, the
earlier developmental study specifically used adult epicardial
myocytes,8 whereas the
present study used the whole ventricle of the adult heart to obtain
a higher yield of viable cells for culture. A gradient of
If
activation, with epicardium more negative than endocardium, has been
observed in the canine
heart.44 If a similar
gradient exists in adult rat ventricle, then this also could contribute
to the less negative adult values observed in the present
study.
Expressing high levels of HCN2 in a neonatal culture results in a marked increase in spontaneous rate. This is accompanied by a less negative maximum diastolic potential and more pronounced phase 4 slope. The maximum diastolic potential in the HCN2-infected culture corresponds to the threshold voltage of IHCN2, indicating that even threshold levels of expressed current are sufficient to balance the contribution of IK1 (which is small in neonatal cultures45 ). Expressing HCN2 in adult myocytes does not result in automaticity, either because of the more negative activation range in the adult cells or the greater IK1 density at this age. However, it does increase the susceptibility to anode break excitation, and this occurs without any alteration of IK1 (data not shown). In HCN2-infected adult cultures, the maximal negative voltage required to exhibit anode break excitation corresponds to the threshold voltage of IHCN2. Thus, the physiological impact of overexpression of the HCN gene family in myocardium depends on the threshold voltage of the expressed current, which is context-dependent, with a distinct result depending on the maturational state of the target tissue. For the same reason, the effect is likely to depend on the cardiac region in which the channel is expressed and the disease state of the tissue, because native If is markedly affected by these factors. This has obvious implications for future efforts to alter cardiac rhythm through the regional overexpression of selective HCN isoforms. It suggests that rate can be enhanced by increasing current level, if the expressed current activates at a physiologically relevant threshold voltage in the target tissue. As we gain further insight into the mechanisms regulating the voltage dependence of this gene family, it may be possible to control both the level of current and its activation voltage and thereby its contribution to an automatic rhythm.
Acknowledgments
This work was supported by NIH grants HL-28958 and NS-36658. Dr Qu is the recipient of a postdoctoral fellowship from the American Heart Association, Heritage Affiliate. We would like to thank Dr Hamish Young for assistance in preparing the adenoviral construct, Drs Michael Rosen and Steven Siegelbaum for advice and suggestions on the manuscript, and Ema Stasko, Hae-Jung Chung, and Patricia McLaughlin for technical assistance.
Footnotes
Original received May 2, 2001; resubmission received June 13, 2001; accepted June 13, 2001.
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