In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionality

J Physiol 587.7 (2009) pp 1513–1525 1513

In vitro characterization of HCN channel kinetics andfrequency dependence in myocytes predicts biologicalpacemaker functionality

Xin Zhao1, Annalisa Bucchi1, Ronit V. Oren3, Yelena Kryukova1, Wen Dun1, Colleen E. Clancy3

and Richard B. Robinson1,2

1Department of Pharmacology and 2Center for Molecular Therapeutics, College of Physicians and Surgeons, Columbia University, New York, NY, USA3Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY, USA

The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN)channels, contributes to the initiation and regulation of cardiac rhythm. Previous experimentscreating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by1–5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytesover-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressingmyocytes manifest similar voltage dependence, current density and sensitivity to saturatingcAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared withHCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence ofirregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore theserhythm differences further, we imposed consecutive pacing and found that activation kineticsof the two channels are slower at faster pacing frequencies. As a result, time-dependent HCNcurrent flowing during diastole decreases for both constructs during a train of stimuli at a rapidfrequency, with the effect more pronounced for HCN2. In addition, the slower deactivationkinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency.As a result of the frequency dependence of both instantaneous and time-dependent current,HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenanceof a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs inspontaneously active myocyte cultures and may provide the basis for future optimization ofHCN-based biological pacemakers.

(Received 16 September 2008; accepted after revision 23 January 2009; first published online 26 January 2009)Corresponding author R. B. Robinson: Center for Molecular Therapeutics, Columbia University, 630 West 168 Street,PH7West-318, New York, NY 10032, USA. Email: [email protected]

The pacemaker current (I f ) plays a major role inthe initiation and regulation of cardiac rhythmdue to its activation upon hyperpolarization andmodulation by direct binding of cAMP (DiFrancesco,1993). The molecular correlates of I f are thehyperpolarization-activated cyclic nucleotide-gated(HCN) channels (Biel et al. 1999; Santoro & Tibbs,1999; Accili et al. 2002). There are four HCN isoforms(HCN1–4) in mammals, and three of these are present inheart. HCN1 shows the fastest kinetics but low sensitivityto intracellular cAMP. Both HCN2 and HCN4 respond

This paper has online supplemental material.

strongly to cAMP, but HCN4 has slower kinetics. Recently,HCN-based biological pacemakers have been reported(Qu et al. 2003; Plotnikov et al. 2004, 2008; Xue et al.2005; Bucchi et al. 2006; Lieu et al. 2008). Because ofHCN2’s strong cAMP response and moderate kinetics,our group has focused on this isoform as the backboneof a biological pacemaker and has made mutant andchimeric channels in an attempt to optimize HCN2-basedbiological pacemakers (Bucchi et al. 2006; Plotnikov et al.2008).

A major focus has been the chimeric channel, HCN212,which incorporates the pore-forming portion of HCN1and the amino- and carboxy-termini of HCN2. Thischimeric channel combines the fast activation kinetics

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society DOI: 10.1113/jphysiol.2008.163444

1514 X. Zhao and others J Physiol 587.7

of HCN1 and the more robust cAMP response ofHCN2 (Wang et al. 2001). However, HCN212 manifestedpacemaker rates in vivo that are too rapid to be desirableand these were interspersed with excessively long pauses.Therefore we embarked on a systematic study to explorewhich biophysical properties contribute to the differencebetween HCN2 and HCN212 channels with respect tocardiac pacing.

HCN channels incorporate a slowly time-dependentcomponent (I HCN) and an instantaneous current (I ins)(Proenza et al. 2002; Proenza & Yellen, 2006). I ins canbe enhanced by incomplete deactivation and stronglydepends on the frequency of hyperpolarizing pulses(Mistrik et al. 2006). In addition, it has been reportedthat the voltage dependence of HCN channels exhibitshysteresis under non-equilibrium conditions (Azeneet al. 2005), and recent allosteric models provide atheoretical framework for this behaviour (DiFrancesco,1999; Altomare et al. 2001; Wang et al. 2002). Duringcardiac activity the membrane potential of the myocytechanges dynamically and always is in a non-equilibriumstate. Thus, contributions of both the instantaneous andtime-dependent components of I f to diastole shouldvary with pacing frequency. We hypothesized that thefrequency dependence of HCN2 and HCN212 mightdiffer, contributing to their distinct functionality asbiological pacemakers in vivo. We therefore have exploredboth the equilibrium and non-equilibrium characteristicsof HCN2 and HCN212 currents. Further, we conductedthese studies in newborn rat ventricular myocytes toprovide a cardiac context, because the expression systemcan affect the functional characteristics of expressedchannels (Qu et al. 2002). This also allowed us to studythe effect of these constructs on spontaneous activity.

Methods

Ethical approval

Animal handling procedures were reviewed and approvedby the local Institutional Animal Care and Use Committeeof the Columbia University Medical Center, and conformto the Guide for the Care and Use of Laboratory Animals(National Institutes of Health publication No. 85-23,revised 1996).

Cell culture

Newborn rat ventricular myocyte cultures were preparedas previously described (Protas & Robinson, 1999). Briefly,1- to 2-day-old Wistar rats (Charles River Laboratories,Wilmington, MA, USA) were killed by decapitation andventricles were dissociated using a standard trypsinizationprocedure. Myocytes were harvested and incubated in

serum-free medium at 37◦C, 5% CO2. Action potentialstudies were conducted on 4 to 6 day monolayer culturesplated directly onto fibronectin-coated 9 mm × 22 mmglass coverslips. For electrophysiology experiments, thecell monolayer was briefly (1–2 min) exposed to 0.1%trypsin, and cells were replated onto fibronectin-coatedcoverslips at low density, and then studied 2–6 h later.

Adenoviral constructs and expression

Both mHCN2 and mHCN212 were packaged inthe pDC515 shuttle vector (AdMax, MicrobixBiosystems, Inc., Toronto, Ontario, Canada), thencotransfected with pBHGfrt�E1, 3FLP vector (AdMax)into E1-complementing HEK 293 cells. Successfulrecombination of the shuttle plasmid and adenoviralgenomic vector resulted in production of the appropriateadenovirus, which was expressed and studied in newbornrat ventricular myocytes as described previously (Qu et al.2001). Since we find that > 90% of cells exposed to anHCN adenovirus (multiplicity of infection 20) in vitroexpress pacemaker currents, we did not co-infect withgreen fluorescent protein (GFP)-expressing adenovirusto identify expressing cells. However, we employed aGFP-expressing adenovirus as our control.

Electrophysiology

The whole-cell patch clamp technique was used to measureHCN currents from newborn rat ventricular myocytesas previously described (Qu et al. 2001). Data wereacquired at 10 kHz using an Axopatch 200B amplifierand pCLAMP 9 (Molecular Devices, Sunnyvale, CA,USA). Voltage clamp data were analysed off-line usingClampfit 9 (Molecular Devices) and Origin 7 (OriginLab Corp., Northampton, MA, USA). All recordingswere obtained at 35◦C. The standard external solutioncontained (in mmol l−1): NaCl, 140; NaOH, 2.3; MgCl2,1; KCl, 10; CaCl2, 1; Hepes, 5; glucose, 10; pH 7.4.MnCl2 (2 mmol l−1) and BaCl2 (2 mmol l−1) were addedto block other currents during whole-cell patch clamprecording. For action potential recording the blockerswere omitted and KCl reduced to 5.4 mmol l−1. Thepipette solution contained (in mmol l−1): aspartic acid,130; KOH, 146; NaCl, 10; CaCl2, 2; EGTA-KOH, 5;Mg-ATP, 2; Hepes-KOH, 10; pH 7.2. The pipetteresistance was typically 2–3 M�. Current densities werenormalized to cell membrane capacitance. Records werenot corrected for liquid junction potential, which was pre-viously determined to be 9.8 mV under these recordingconditions.

Action potential (AP) recordings were done inwhole-cell mode on a monolayer culture. Action potentialparameters were measured on four successive APs and

C© 2009 The Authors. Journal compilation C© 2009 The Physiological Society

J Physiol 587.7 HCN channel frequency dependence and automaticity 1515

averaged to determine the values for each culture plate.We measured maximum diastolic potential (MDP),take-off potential (TOP) and early diastolic depolarization(EDD), all as previously described (Bucchi et al. 2007).Spontaneous rate was determined as the reciprocal of theaverage cycle length over a 2 s period.

Whole-cell currents were evoked with the followingprotocols. Protocol 1: To measure the HCN activationrelation and activation time constant, hyperpolarizingsteps from −25 to −125 mV were applied from a holdingpotential of −25 mV, followed by a tail current step to−85 mV. The duration of test steps was 3 s. Protocol 2:To measure HCN deactivation, currents were recordedfor 5 s with a family of test voltages ranging from −80to +20 mV preceded by a 1 s prepulse to −120 mV.Protocol 3: We performed an envelope test to illustratethe effect of interpulse interval on HCN channels for avoltage corresponding to typical action potential over-shoot. The membrane was stepped from the −30 mVholding potential to −80 mV for 4 s, and then steppedto +20 mV for a variable time interval, after which it wasreturned to −80 mV for 4 s. Test pulses were repeatedevery 12 s. Protocol 4: To analyse the effect of the lengthof a hyperpolarizing prepulse on the activation kineticsof HCN channels, the membrane was stepped fromthe −30 mV holding potential to −80 mV for differentdurations and then returned to +20 mV for 150 ms(to approximate typical action potential duration) andstepped again to −80 mV for 6 s. Test pulses were repeatedevery 12 s. Protocol 5: To explore the effect of consecutivestimuli on HCN channels, a train of square pulses wasused to mimic action potentials. The holding potentialwas −80 mV for a duration long enough (4–6 s) to achieveequilibrium. Subsequently, two protocols were performedby consecutively stepping to +20 mV for 150 ms followedby a step to −80 mV for 150 or 1850 ms to mimic cyclelengths of 300 and 2000 ms, respectively. To minimize theeffect of current rundown or drift, the order of the twoprotocols was alternated in successive cells.

For activation curves, the normalized plot of tail currentversus test voltage was fitted to a Boltzmann equation:

I = A 1 + A 2/(1 + exp[(V − V1/2)/s])

in which A1 is the offset of the holding current, A2 is themaximal tail current amplitude, V is the voltage during thehyperpolarizing test pulse, V 1/2 is the midpoint potentialof activation, and s is the slope factor. The normalized datawere averaged among different experiments and refittedto the Boltzmann equation with A1 = 0 and A2 = 1.Kinetics of activation (τact) were determined from thesame tracings by a single exponential fit to the early timecourse of the current activated by hyperpolarization. Theinitial delay and any late slow activation were ignored.Kinetics of deactivation (τdeact) were determined by a

single-exponential fit of the time course of the currenttrace at each test voltage after activation by a prepulseto −120 mV. For estimating open and close rates, thebell-shaped distribution of averaged τact and τdeact wasfitted to:

τ = 1/(αe−Vt/V0 + βeVt/V0 )

where α and β reflect the open and close rates at zerovoltage, respectively, V t is the test voltage, and V 0 isthe voltage at which the rate constants change by e-fold.To obtain information about the biological behaviour ofHCN channels under consecutive stimuli, we measuredthe total I ins and the magnitude of the time-dependentHCN current at the 150 ms time point (I HCN,150 ms). Thisreflects current that would flow during early diastole.

Computer simulation

We employed the sinoatrial node (SAN) model of Kurataet al. (2002), and fitted models to the experimentallymeasured activation/deactivation kinetics of expressedHCN2 and HCN212 in the myocyte cultures (see Table 1and Fig. S1 in the online Supplemental material). The mid-point and slope of the activation curve were also modifiedto match the experimental data. Since the maximumconductance of pacemaker current in the model wascomparable to that of both HCN2 and HCN212 in thecultures, we did not modify that parameter.

Statistical analysis

Pooled data are presented as mean ± S.E.M.; n denotesthe number of cells. To minimize the effect ofculture-to-culture variability, for each culture cells wereprepared expressing each construct and data from at leastthree separate cultures were pooled for each comparison.Student’s paired t test or Fisher’s exact test was performedfor comparison between two groups, as appropriate. AnANOVA was performed for multiple comparison, followedby a modified t test with Bonferroni correction (SigmaStat,Systat Software Inc., San Jose, CA, USA). Values ofP < 0.05 were considered significant.

Results

Biophysical properties of HCN2 and HCN212 currentsexpressed in newborn rat ventricular myocytes

Because the expression level of HCN channels caninfluence the effect on cardiac pacing (Bucchi et al. 2006),we first compared the expression efficiency of HCN2 andHCN212 channels in myocytes. There was no difference incell capacitance between HCN2- and HCN212-expressingmyocytes (30.0 ± 2.2 pF, n = 26, and 33.0 ± 3.2 pF, n = 22,



respectively; P > 0.05). In whole-cell voltage clampexperiments, both HCN2- and HCN212-expressing myo-cytes gave rise to robust inward currents in response tohyperpolarizing voltages. The HCN212 chimeric channelswere well expressed in newborn rat ventricular myo-cytes, similar to our findings for HCN2 channels (Quet al. 2001). The percentage of myocytes expressingHCN212 current was not significantly different fromthat of myocytes expressing HCN2 current (91% and93% for HCN2 and HCN212, defined as percentageof cells with pacemaker current > 200 pA at −115 mV;n = 33 and 27, respectively). Representative normalizedcurrent traces are shown in Fig. 1A. For I HCN, the currentdensity in the two groups at any voltage was not different(Fig. 1B). For example, mean current densities of I HCN2

and I HCN212 at −125 mV were 29.0 ± 4.5 pA pF−1 (n = 8)and 33.9 ± 9.8 pA pF−1 (n = 12, P > 0.05), respectively.

Figure 1. Expression of HCN2 and HCN212 in newborn rat ventricular myocytesA, representative whole-cell current tracings of myocytes expressing HCN2 (top) and HCN212 (bottom) channels.Currents were evoked by stepping from a holding potential of −25 mV to different hyperpolarizing voltage stepsranging from −25 to −125 mV in 10 mV increments. B, the mean I–V relationship of HCN2 (n = 8, filled circles)and HCN212 (n = 12, open circles). The HCN current (IHCN) is defined as the time-dependent component at theend of the 3 s test pulse. C, representative instantaneous current I ins,HCN2, I ins,HCN212 and I ins,GFP recorded at asingle hyperpolarizing step (−120 mV) from a holding potential of −30 mV. The inset shows how the instantaneouscomponent was measured; it is defined as the difference between the zero current level and the initial current levelwhere the time-dependent current component begins, and includes leak current. D, the instantaneous currentdensity in response to voltage steps to −120 mV in myocytes over-expressing HCN2 (n = 15), HCN212 (n = 13),GFP (n = 17). ∗P < 0.05 versus GFP.

We also examined the magnitude of the instantaneouscurrent (I ins) of HCN channels, defined as the differencebetween the zero current level and the initial currentfollowing a voltage step. Since we were interested indifferences in this component between constructs (andas a function of frequency, as described below), ratherthan absolute value, we did not conduct experiments tomeasure and subtract leak current from these records.As shown in Fig. 1C and D, the mean current densitiesof I ins,HCN2 and I ins,HCN212 using a single step from−30 mV to −120 mV were 8.2 ± 1.0 pA pF−1 (n = 15)and 6.8 ± 0.7 pA pF−1 (n = 13, P > 0.05), respectively,and both values were larger than that in GFP-expressingmyocytes (3.7 ± 0.6 pA pF−1, n = 17, P < 0.001). Theseobservations indicate that HCN2 and HCN212 channelexpression is comparable in newborn rat ventricular myo-cytes and that, under these recording conditions, the



two constructs produce equivalent instantaneous andsteady-state time-dependent current.

We further analysed the voltage dependence of HCN2and HCN212 channels heterologously expressed in myo-cytes under equilibrium recording conditions. HCN2 andHCN212 channels exhibited similar voltage dependenceof activation as shown in Fig. 2A. The mean V 1/2 was−65.9 ± 1.4 mV (n = 8) for HCN2 versus −65.2 ± 1.9 mV(n = 10) for HCN212 (P > 0.05). The corresponding svalues were 8.2 ± 0.9 mV and 10.7 ± 1.1 mV respectively(P > 0.05). The similar voltage dependence of HCN2 andHCN212 in newborn myocytes is consistent with dataobtained previously in adult myocytes (Plotnikov et al.2008).

An important criterion of an HCN gene-basedbiological pacemaker is better autonomic responsiveness,resulting from direct binding of cAMP to HCNchannels (Bucchi et al. 2006; Robinson et al. 2006).To test whether HCN2 and HCN212 expressedin myocytes exhibited similar cAMP responsiveness,the activation curves were further investigated inthe presence of saturating cAMP in the pipettesolution (10 μmol l−1). Both channels responded equallyto the presence of saturating intracellular cAMP.In the presence of cAMP, calculated values wereV 1/2 = −56.4 ± 1.2 mV and s = 7.8 ± 0.6 mV for HCN2(n = 6); V 1/2 = −56.9 ± 2.7 mV and s = 9.9 ± 1.1 mV forHCN212 (n = 7), respectively. For HCN2 and HCN212,control and cAMP relations differ significantly (a 9.5 mV

Figure 2. Steady-state properties of HCN2 and HCN212 in newborn rat ventricular myocytesA, the mean activation–voltage relation of HCN2 and HCN212 in the absence and presence of cAMP. Meanfractional activation curves of HCN2 (squares) and HCN212 (circles) obtained in the absence (black symbols) and inthe presence (grey symbols) of 10 μmol l−1 cAMP in the pipette solution. For illustrative purposes, the mean datawere fitted to the Boltzmann equation for experiments in the absence (continuous black lines) and in the presence(dashed grey lines) of cAMP. Inset: mean V1/2

values without (black) and with (grey) cAMP. B, voltage dependenceof activation (filled symbols) and deactivation (open symbols) time constants of HCN2 (squares) and HCN212(circles). Mean activation values were obtained from 10 cells for both HCN2 and HCN212; mean deactivation timeconstant values were obtained from 7 and 6 cells for HCN2 and HCN212, respectively. The curves are the fits tothe equation τ = 1/(αe−Vt/V0 + βeVt/V0 ). ∗Voltages differ by multiple comparison (P < 0.05).

and 8.3 mV shift, respectively). HCN2 and HCN212curves do not differ from each other under either controlconditions or in the presence of cAMP (P > 0.05).

We next analysed the gating kinetics of HCN channelsin myocytes. The representative currents demonstratedthat HCN212 channels exhibit faster activation kineticsthan HCN2 (Fig. 1A). In addition, when membranepotential was depolarized after stepping to −120 mV tofully activate HCN212 channels, the deactivation kineticsof HCN212 channels were also faster. The completerelationship between gating kinetics (both activation anddeactivation) and membrane potential is illustrated inFig. 2B for HCN212 and HCN2. Plotting these timeconstants together against the test potential revealed thatthe voltage dependence of τact and τdeact had a bell-shapedform. α and β derived from this curve were: 1.2 × 10−4

and 0.03498 for HCN212; 3 × 10−5 and 0.01598 forHCN2. Analysis of gating kinetics indicate that the meanactivation and deactivation time constants of HCN212channels are faster than those of HCN2 channels overmost of the voltage range analysed, and the relative peaksof the kinetics–voltage relationships are consistent withthe previously determined V 1/2 values.

Thus, under equilibrium conditions HCN2 andHCN212 channels expressed in newborn rat ventricularmyocytes have equivalent voltage dependence, currentamplitude and responsiveness to saturating cAMP, butHCN212 exhibits faster activation and deactivationkinetics.



Computer simulation of effect of HCN kineticson automaticity

To investigate the possible impact of the difference inactivation and deactivation kinetics on spontaneous rate,we utilized the Kurata computer model of the SAN(Kurata et al. 2002). Supplemental Fig. S1 shows thevoltage dependence of activation gating (panel A) andactivation/deactivation time constants (panel B) in theoriginal SAN model and after modification to matchexperimental data in myocytes expressing HCN2 orHCN212 (as shown in Fig. 2). Compared to I f in theoriginal SAN model, expressed HCN2 activation kineticsare faster at negative potentials, but at less negativepotentials within the diastolic potential range, activationkinetics are slower in HCN2 channels. In addition,deactivation kinetics are slower at all voltages. HCN212has faster activation kinetics than SAN I f at all voltages,while at potentials positive to −40 mV the deactivationkinetics of HCN212 and SAN I f are comparable.

The resulting families of simulated currents in eachcase are shown in Fig. 3A. Conductance was the samein all three simulations, and the current magnitude forHCN2 and HCN212 in the simulation match the meanexperimental values (Fig. 1B). Simulated spontaneousaction potentials are shown in Fig. 3B. Using the HCN2experimental data, the cycle length was longer than thatof the SAN model (329 versus 308 ms), reflecting aslower rate as a result of the slower activation kinetics

Figure 3. Simulated whole-cell current tracing and simulated spontaneous action potentials in theKurata sinoatrial node (SAN) model with original Kurata If, HCN2 expression and HCN212 expressionA, currents were evoked by stepping from a holding potential of −25 mV to different hyperpolarizing voltagesteps ranging from −25 to −125 mV in 10 mV increments. B, spontaneous action potentials were simulated witheach of the pacemaker current configurations – the SAN model with unchanged If (Kurata et al. 2002) is shownon the left, HCN2 in the middle, and HCN212 on the right.

at diastolic potentials. When the HCN2 parameters arereplaced by those of HCN212, cycle length shortens (from329 to 305 ms). To determine if this was entirely dueto the faster activation, we also undertook a simulationwhere we changed only the activation kinetics to matchthe HCN212 data, leaving deactivation and all otherparameters unchanged. In this case cycle length was alsoshorter than in the pure HCN2 simulation (318 ms),but not as short as in the full HCN212 simulation.Thus both activation and deactivation kinetics can impactspontaneous rate.

The effect of HCN2 and HCN212 expressionon spontaneous beating of newborn myocytes

The computer simulations lend validation to thehypothesis that changes to channel kinetics can resultin changes in spontaneous rate. We therefore nextexplored the effect of the two HCN constructs onspontaneous activity of monolayer cultures of newbornrat ventricular myocytes. Spontaneous action potentials(APs) were recorded from a myocyte within the mono-layer using a whole-cell patch electrode. As expectedfrom the faster activation kinetics with comparableexpression efficiency, and the computer simulation,HCN212 resulted in a faster spontaneous rate thanHCN2 channels (234 ± 10 beats min−1, n = 25, and



177 ± 12 beats min−1, n = 26, respectively, P < 0.05), andboth groups beat faster than did control culturesexpressing GFP (116 ± 19 beats min−1, n = 7, P < 0.05,Fig. 4A).

However, the incidence of bursting behaviour inHCN212-expressing monolayers was significantly greaterthan that in HCN2 cultures; 40% (10 out of 25) and 11.5%(3 out of 26 cells, P < 0.05, Fig. 4B and C), respectively. Inboth groups, the rate within the burst was statistically notdifferent from the rate observed in non-bursting cultures(HCN212: 228 ± 16 versus 238 ± 19, P > 0.05; HCN2:147 ± 24 versus 181 ± 13, P > 0.05, respectively).

We considered that the difference in rhythmicity couldbe secondary to the effects of one of the constructs onother ionic currents. We first explored this by measuringaction potential parameters in each case. The HCN2and HCN212 cultures (n = 25 each) did not differwith respect to action potential amplitude (94.74 ± 2.24and 96.76 ± 2.24 mV, respectively; P > 0.05), MDP(−58.76 ± 1.19 and −59.55 ± 1.33 mV, respectively;P > 0.05) or TOP (−51.38 ± 1.32 and −49.31 ± 1.62 mV;P > 0.05), but EDD was significantly greater with HCN212

Figure 4. Effects of HCN2 and HCN212 channel expression on spontaneous activity of newborn ratventricular myocytesA, mean spontaneous beat rate of myocytes expressing GFP, HCN2 and HCN212. ∗P < 0.05 versus GFP; ∗∗P < 0.05versus HCN2 and GFP. B, representative examples of spontaneous action potentials with bursting behaviourrecorded from an HCN2-expressing (top) and HCN212-expressing culture (bottom). At the far right, the portionof each trace indicated by the box is displayed on an expanded time scale. C, the incidence of bursting behaviourin HCN-expressing monolayers. ∗P < 0.05 versus HCN212.

expression, consistent with the higher rate (0.06 ± 0.01and 0.14 ± 0.01 V s−1, respectively; P < 0.05). Regressionanalysis indicated that any differences in actionpotential duration were secondary to differences inrate (Supplemental Fig. S2). We also investigated othercurrents that could contribute to excitability and/orautomaticity. We measured the inward rectifier current,defined as the current blocked by 1 mM Ba2+. Theseexperiments were done in the presence of 3 × 10−6 mol l−1

ivabradine (Servier Laboratories, Courbevoie, France)to minimize any possible contamination from the largeHCN currents. At −80 mV the current magnitudes were89 ± 19 and 107 ± 22 pA (n = 7–8; P > 0.05) for HCN2and HCN212, respectively. We also measured inward Na+

current, and found no difference in the peak current,the I–V relation, or the inactivation relation in HCN2-and HCN212-expressing cultures (Supplemental Fig. S3).Finally, we performed Western blot analysis to determineprotein expression in HCN2 versus HCN212 culturesof Na+ channels (using a pan-Na+ antibody) and theKir2.1 component of the inward rectifier; expression wascomparable in both cases (Supplemental Fig. S4A). We



did find HCN212 expression was greater than that ofHCN2; however, when the light sarcolemmal membranefraction was analysed separately expression was equivalent(Supplemental Fig. S4B).

Thus, neither the difference in spontaneous rate northe irregularity of rhythm observed with HCN212 canreadily be ascribed to secondary effects of one constructon other ionic currents. While the computer simulationsuggests that the faster rate can be explained by thealtered kinetics, it should be noted that the observationof irregular rhythms with HCN212 was not observed inthe simulation, which is based on a Hodgkin–Huxleyformulism of the pacemaker channel. Such a modeldoes not replicate the non-equilibrium behaviour ofthe channel, which requires more complex allostericmodelling. This therefore led us to experimentally explorethe non-equilibrium behaviour of the two constructs ingreater detail.

Modulation of HCN channels under non-equilibriumconditions

At faster spontaneous rates, myocytes spend more timein a depolarized state. To explore the impact of this onboth the instantaneous and time-dependent componentof HCN current, we imposed two different double hyper-polarizing protocols. First, we performed protocol 3 (anenvelope test, see Methods) with varying interpulse inter-vals for a step to +20 mV. This was intended to illustratethe effect of AP duration (APD; mimicked with a squarepulse to +20 mV of varying duration) on residual I ins.Representative tracings of HCN2 evoked by the protocolare shown in Fig. 5A. As expected, there is a reciprocalrelation between the instantaneous and time-dependentcomponents of the current, with the time-dependentcomponent of HCN current (I HCN) becoming smalleras the interpulse interval decreases. The correspondingenhancement of I ins (�I ins) reflects residual current fromincomplete deactivation of HCN channels as a resultof the short time at depolarized potentials (Proenza &Yellen, 2006). Plotting �I ins against the interpulse inter-val (Fig. 5B) yields the average time course of currentdeactivation for the test pulse of +20 mV. An exponentialfit to these data yields time constants of 52.8 ± 8.3 ms(n = 4) and 21.7 ± 3.3 ms (n = 3) for �I ins,HCN2 and�I ins,HCN212, respectively (P < 0.01). As illustrated by thedashed vertical line at 150 ms, for HCN2 the increase ininstantaneous current equals 8.4% of the time-dependentcurrent, while for HCN212 the increase at this timerepresents only 0.2% of the time-dependent current,reflecting the more rapid deactivation of HCN212. Thus,for a typical APD, significantly more HCN2 channels arelikely to remain open at the start of diastole compared toHCN212 channels. While the former protocol exploredI ins, the next protocol looked at the effect of voltage

history on the time-dependent current (protocol 4, seeMethods) by imposing varying prepulse durations. The+20 mV interpulse was fixed at 150 ms and the pre-pulse duration (to a voltage of −80 mV) varied between150 ms and 2700 ms, in 850 ms increments (Fig. 5C andD). HCN2 channels at the shortest, 150 ms prepulseduration, corresponding to a short diastolic interval,exhibited significantly slower activation kinetics thanfor the other durations (P < 0.05). Similar to HCN2channels, in HCN212 channels we also found a significantdifference in the activation time constant at the 150 msprepulse duration (P < 0.01). These results demonstratethat HCN2 and HCN212 channels expressed in myo-cytes display a prepulse-dependent activity as reportedpreviously for other HCN channels (Wang et al. 2002;Proenza & Yellen, 2006).

Taken together, Fig. 5D illustrates that for HCNchannels both the magnitude of instantaneous andactivation kinetics of the time-dependent current can varyfor physiologically relevant APDs and diastolic intervals,and that HCN2 and HCN212 may quantitatively differ inthis regard. We therefore addressed frequency-dependentmodulation of HCN channel activity more directly,using two different protocols to approximate the timecourse of membrane potential changes in a beatingmyocyte at heart rates of 0.5 Hz (30 beats min−1) and3.3 Hz (200 beats min−1), respectively. Figure 6A showsrepresentative HCN currents elicited by a protocol witha depolarizing step to +20 mV lasting 150 ms from aholding potential of −80 mV followed by continuouslyrepeating 20 square voltage pulses at 0.5 Hz. We comparedthese results to those obtained at 3.3 Hz. HCN currentsvary at the two frequencies, particularly for HCN2,as seen when the final voltage steps are superimposedin Fig. 6B. Next, we examined the total instantaneouscurrent (I ins). As shown in Fig. 6C, the magnitudeof I ins of HCN2 channels following the 20th pulseat 0.5 Hz was greater than that at 3.3 Hz (8.3 ± 1.5and 4.4 ± 1.2 pA pF−1, n = 8, P < 0.01). By contrast, themagnitude of I ins of HCN212 channels remained smalland constant at 3.3 Hz and 0.5 Hz, 4.4 ± 0.6 pA pF−1 and4.0 ± 0.7 pA pF−1 (n = 6, P > 0.05), respectively. Thus,compared with HCN212, HCN2 exhibits a large I ins at0.5 Hz, presumably because a slower rate allows morecurrent to activate during the period at −80 mV, andthus results in more current remaining as a consequenceof incomplete deactivation during the pulse to +20 mV.Second, after 20 consecutive square voltage pulses followedby stepping back to −80 mV, the time constant ofchannel activation was measured by fitting to a singleexponential function. As shown in Fig. 6D, both HCN2and HCN212 channels at 0.5 Hz exhibit faster activationkinetics (smaller τ values) than at 3.3 Hz (for HCN2:387 ± 44 ms to 483 ± 37 ms, P < 0.05; for HCN212:118 ± 6 ms to 160 ± 9 ms, P < 0.05). In addition, as



evident in Fig. 6B, the HCN2 delay prior to activationof the time-dependent component is decreased at 0.5 Hz(22.3 ± 1.6 ms versus 29.3 ± 2.6 ms at 3.3 Hz, P < 0.05).To assess the impact of the slower activation and longerdelay at faster rates, we measured the magnitude ofthe time-dependent component of HCN currents at the150 ms time point (I HCN,150 ms) following each of the20 step pulses (Fig. 6E). The current contribution ofboth channels remained relatively stable throughout thestimulus train at 0.5 Hz, normalized to I HCN,150 ms of thefirst stimulus. By contrast, the currents of HCN2 andHCN212 significantly decreased at 3.3 Hz. However, thereduction in HCN2 was significantly greater than that

Figure 5. Prepulse modulation of HCN channelsA, representative HCN2 currents elicited by protocol 3. The inset shows the entire protocol, which includes anactivating prepulse to −80 mV followed by a step to +20 mV with increasing interpulse interval and an additionalactivating step to −80 mV; the portion of the protocol not displayed in the current traces is represented bydashed lines. The double arrow labelled I ins represents the constant portion, or minimal, I ins. The double arrowlabelled �I ins indicates the enhancement of the instantaneous current observed at the shortest interpulse interval,reflecting the largest measured �I ins. IHCN is the time-dependent steady-state current, and its magnitude is labelledfor the same current trace. B, dependence of �I ins,HCN2 (n = 3, squares) and �I ins,HCN212 (n = 3, circles) on theinterpulse interval. �I ins was normalized to the amplitude of the time-dependent steady-state component of HCNcurrent (�I ins/IHCN). Data were fitted to a single exponential function; the 150 ms time point is highlighted by thevertical dashed line. C, representive HCN2 currents elicited by protocol 4. The inset shows the full protocol, withthe portion omitted from the current traces represented by dashed lines. Prepulses (−80 mV) were evoked fordifferent prepulse lengths (150 ms, 1000 ms, 1850 ms and 2700 ms) and the interpulse interval kept at 150 ms.D, activation time constant of HCN2 (n = 5, squares) and HCN212 (n = 4, circles) during the second −80 mVstep. ∗Statistically significant (P < 0.05) difference from other prepulse durations in same HCN group by multiplecomparison.

in HCN212, beginning from the 8th stimulus (P < 0.05).These results indicate that the time-dependent componentof HCN current present early in diastole decreases at fasterpacing, and does so more markedly for HCN2 than forHCN212.

Discussion

We have over-expressed HCN channels in newborn ratventricular myocytes with the aim of investigating whichcharacteristics mainly account for the different biologicalpacemaker behaviour of HCN2 and HCN212 channels invivo. The results show that: (1) both HCN2 and HCN212



channels expressed in myocytes have similar values ofvoltage dependence, current density and sensitivity tosaturating cAMP, but HCN212 has faster activation anddeactivation kinetics; (2) compared with HCN2, myo-cytes expressing HCN212 have a greater likelihood ofexhibiting a faster but less regular rhythm, comparableto what is observed in vivo (Plotnikov et al. 2008); and (3)under non-equilibrium conditions, HCN2 and HCN212exhibit different behaviour due to their different gatingcharacteristics.

The pacemaker current is thought to play a role ingenerating and regulating pacemaker activity, but itsrelative importance is still controversial (DiFrancesco,1993; Lakatta et al. 2003). However, when over-expressedsuch that a large current is generated in the diastolicpotential range, there is now abundant evidence thatthe HCN gene family can create biological pacemakeractivity (Plotnikov et al. 2004, 2008; Xue et al. 2005;

Figure 6. Frequency-dependent modulation of HCN currents in myocytesA, representative HCN currents elicited by repetitive steps from −80 mV to +20 mV for 150 ms at 0.5 Hz pacingrates. B, overlapping of the 20th HCN2 current (top) and HCN212 current (bottom) at 0.5 (black) and 3.3 Hz(grey) pacing rates. C, the changes of total I ins in HCN2 (n = 8) and HCN212 (n = 6) at 0.5 (white) and 3.3 Hz(black). ∗P < 0.05 versus 3.3 Hz by paired test; #P < 0.05 versus HCN212. D, activation time constant of HCN2and HCN212 during the last −80 mV step at 0.5 (white) and 3.3 Hz (black) pacing rates. ∗P < 0.05 versus 3.3 Hzby paired test. E, the mean beat-to-beat changes of the time-dependent current of HCN2 (squares) and HCN212(circles) at the 150 ms time point (IHCN,150 ms) during the 20-pulse train at 0.5 Hz (open symbols) and 3.3 Hz (filledsymbols). ∗P < 0.05 versus HCN212 at 3.3 Hz by multiple comparison.

Bucchi et al. 2006). The inward current contributed byI f in the diastolic range of potentials is determined bythe number of HCN channels opened during diastolicdepolarization, which in turn depends on the steady-stateopen probability at a given potential and the rate at whichthe channel gating variable approaches the steady-statevalue (Bucchi et al. 2006). Under equilibrium conditions,HCN2 and HCN212 channels expressed in newborn ratventricular myocytes have similar voltage dependence,current amplitude and responsiveness to saturating cAMP,but HCN212 exhibits the faster kinetics of HCN1, whichhave been ascribed, based on an allosteric model, to highervoltage sensor rates and to closed/open transitions havinglooser voltage-independent interactions (Altomare et al.2001). HCN212 expression accelerated the spontaneousrate of newborn myocytes more than HCN2, which wouldbe consistent with faster current kinetics as confirmedby the computer simulation. The slower deactivation



kinetics of HCN2 contributes to the greater instantaneouscurrent observed at slower rates immediately followinga depolarizing/repolarizing step. That this did not resultin a less negative MDP than in the case of HCN212expression suggests that it is largely offset by the morerapid activation of HCN212. It should be noted that thecomputer simulation fits the macroscopic deactivationkinetics but does not account for the additional dynamicrate-dependent component, and so was not used to explorethe basis of rate irregularity.

Interestingly, the irregular rhythm of rapid rates inter-spersed with pauses, observed with HCN212 in vivo, wasalso evident in the myocyte cultures. To gain insightinto the mechanism underlying the different behaviourof HCN2 and HCN212 in myocytes, we activated thechannels using voltage protocols designed to approximatethe dynamic conditions that occur during spontaneousactivity. As expected, we found that HCN channels weremodulated by pacing frequency, but also that there werequantitative differences between HCN2 and HCN212.

If we first consider I ins, we notice that for depolarizationperiods comparable to typical APD, HCN2 will generate agreater instantaneous current than HCN212 at slow rates,but that the magnitude of I ins will decrease significantlyas spontaneous rate increases (Figs 5B and 6C). In thecase of HCN2, this would provide a form of negativefeedback where the amount of I ins at the beginningof diastole will decrease as rate increases. Consistentwith this interpretation, some studies have demonstratedthat the current from incomplete deactivation of HCNchannels might contribute to the stabilization of pacingrhythm. Analysis of slowly activating HCN4 channelssuggested that enhancement of I ins could be facilitated byincomplete deactivation (Mistrik et al. 2006). The deletionof HCN4 in adult mice eliminates most of sinoatrialI f and results in a cardiac arrhythmia characterized byrecurrent sinus pauses similar to bursting behaviour in ourstudy. Based on the results from a temporally controlledknockout of HCN4, I f was proposed to function as abackup mechanism which guarantees stable pacemaking(Herrmann et al. 2007). However, since both activationand deactivation kinetics of HCN212 differ from those ofHCN2, we cannot rule out a role of rapid activation in theobserved rhythm irregularities.

In addition, the activation kinetics of the two HCNchannels were dependent on the length of the hyper-polarizing prepulse (Figs 5C and D, and 6B and D).Compared to slow continuous pacing (long prepulseduration), a significant decrease in the activation kineticsof HCN channels at fast pacing (short prepulse duration)was observed. Further, as previously reported for nativepacemaker current (DiFrancesco, 1984), the delay shortensat slower rates (i.e. for a longer negative prepulse), whichcan both add to the instantaneous component and resultin a greater time-dependent component at a fixed but

short time point during diastole. As a result of theslower activation and increased delay with faster rates,during a series of 20 simulated action potentials at 3.3 Hz,the time-dependent component of I HCN2 (measured at150 ms) decreased significantly more than that of I HCN212.This would again be consistent with a more robust negativefeedback mechanism for HCN2 than HCN212. Thus,under consecutive pacing at a rapid rate, the negativefeedback characteristics of both the instantaneous andtime-dependent components of HCN2 channels mayfavour the maintenance of a stable rhythm. For HCN212,where the kinetics make it less likely for incompletedeactivation to occur, and where activation kinetics are lessaffected by frequency over the range studied, the negativefeedback is less robust. As a result, if spontaneous rateincreases for any reason, the exogenous pacemaker currentis less able to compensate and restore a slower rate. Ifthe rate becomes fast enough, other mechanisms such asoverdrive suppression may come into play, leading to theobserved pauses and rhythm irregularities.

Our results provided new insight for designing futureHCN constructs for the purpose of creating a biologicalpacemaker. Myocytes expressing HCN212 with fastactivation kinetics have a faster spontaneous rate, whichmay be a desirable feature. However, the slower kineticsof HCN2 channels means that both the instantaneousand time-dependent components of the current exhibitgreater frequency dependence (within the 0.5–3.3 Hzrange studied), which serves to stabilize rhythmicity.By this reasoning, an HCN construct with intermediatekinetics between HCN2 and HCN212 might strike a morefavourable balance. Alternatively, it might prove fruitful touncouple activation and deactivation kinetics, such that aconstruct exhibits the rapid activation kinetics of HCN212but the slower deactivation kinetics of HCN2. While theseparation of activation and deactivation kinetics is notclear-cut, allosteric models suggest they are separableparameters (DiFrancesco, 1999), and detailed insight intothe structure and function of HCN channels may providethe required information in the future. For example,using chimeras between HCN2 and HCN4, it was recentlyreported that the S1–S2 region plays a crucial role indeactivation kinetics (Proenza & Yellen, 2006).

In summary, we propose that frequency-dependentmodulation of HCN channels with distinct activation/deactivation kinetics contributes, at least in part,to the different behaviour of HCN2 and HCN212observed during in vivo expression. The dynamic,or non-equilibrium, characteristics of HCN constructsshould be considered in developing a future HCN-basedbiological pacemaker. Spontaneously beating cultures ofnewborn myocytes represent a useful model system forboth characterizing the biophysical properties of newconstructs and for assessing the effect of these constructson both rate and rhythm.



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Acknowledgements

We thank Drs Dario DiFrancesco, Ira Cohen and MichaelRosen for helpful comments on the manuscript. We thankMs Haejung Chung and Ms Ming Chen for technical support.This study was supported by the National Heart, Lung, andBlood Institute program project grant HL-28958, and theBoston Scientific Corp. X. Zhao was partially supported by the



Specialized Research Fund for the “135” Project (RC2003019)from Jiangsu Province of China.

Author’s present address

X. Zhao: Division of Cardiology, The first affiliated hospital ofSoochow University, Su Zhou, China.

Supplemental material

Online supplemental material for this paper can be accessed at:http://jp.physoc.org/cgi/content/full/jphysiol.2008.163444v1/DC1


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In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionality