Adenoviral Expression of IKs Contributes to Wavebreak and Fibrillatory Conduction in Neonatal Rat Ventricular Cardiomyocyte Monolayers
Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K+ current (IKs) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of IKs in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of IKs) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in IKs-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the IKs group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the IKs reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that IKs-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.
During ventricular and atrial fibrillation, conduction of the electrical wavefront is characterized by complex patterns of propagation, including reentry, wavefront fragmentation (wavebreak), and wavelet formation.1 A wavebreak occurs if the stimulatory efficacy of a wavefront does not suffice to excite all the tissue downstream. The free shoulder of a broken wave is then prone to curl and give rise to a rotor.2 To date, the molecular mechanisms of wavebreaks leading to fibrillatory conduction remain poorly understood.
Studies in single guinea pig myocytes showed that slow recovery of excitability during diastole was, in part, a consequence of the slow gating kinetics of the delayed rectifier potassium outward current IK.3,4 Shortly after these studies were published, IK was found to be the result of the activation of 2 outward currents: IKr and the IKs.5 Given the large amounts of IKs present in guinea pig myocytes6,7 and the slow deactivation kinetics of this current,8 we surmise that IKs is a likely candidate to have an important role in regulating excitability during the diastolic interval, ie, postrepolarization refractoriness.
It is our hypothesis that an increase in the density of IKs may result in enhanced intermittent conduction block and wavebreak formation by means of postrepolarization refractoriness, which may explain fibrillatory conduction in conditions where the IKs current is enhanced, such as during ischemic catecholaminergic release9 or in the case of genetic mutations such as SQT2.10,11
In this study we used cultured neonatal rat ventricular myocyte monolayers infected with an adenovirus carrying the cDNA of the molecular correlate of IKs as a model to investigate the phenomenon of fibrillatory conduction at the molecular level.
Materials and Methods
Myocyte Isolation and Culture
Ventricular myocytes from neonatal Sprague-Dawley rats (Charles River, Mass) were isolated and cultured according to Rohr et al.12 Briefly, the hearts from 1- and 2- day-old rats were aseptically removed and collected in calcium- and magnesium-free Hanks’ Balanced Salt Solution (HBSS). The ventricles were minced and incubated in a solution containing 0.125% trypsin (Roche Applied Science) and 0.15% pancreatin (Sigma). Digestion took place at 36°C in consecutive steps. Two hour differential preplating was used to reduce the presence of noncardiomyocytes. Cells were then suspended in medium M199 (Cambrex) containing 10% fetal bovine serum (FBS) (Cellgro), 20 U/mL penicillin, 20 μg/mL streptomycin, 15 μmol/L Vitamin B12, and 100 μmol/L bromodeoxyuridine (Sigma) to inhibit fibroblast proliferation. Cells were plated on 35-mm tissue culture dishes at a density of 1.2×106 cells/dish for monolayers and at low density in 22-mm coverslips for single cell experiments. Media changes were performed after 24 hours and every 48 hours thereafter with 5% FBS medium. Viral infection was performed at day 3 in culture and myocytes were incubated an additional 24 to 48 hours before experiments to allow for protein expression.
The IKs channel is formed by the coassembly of the pore forming subunit KvLQT1 (KCNQ1) and the regulatory β subunit minK (KCNE1).13,14 We generated an adenoviral construct containing a fused cDNA sequence of human KvLQT1 and minK (Ad_IKs) generously provided by Dr R. Kass (Columbia University).15 The cDNA was subcloned into the AdenoX Adenoviral Expression System (Clontech). Once purified (Virus Purification Kit, Clontech) and tittered (Rapid Titer Kit, Clontech), multicellular preparations were infected with varying multiplicities of infection (MOI) and immunostained using a specific anti-KvLQT1 antibody (US Biological). An MOI of 5 was found to be optimal in terms of uniformity and level of protein expression (Figure 1A). In addition, single neonatal rat ventricular myocytes were infected and standard patch-clamping was used to verify functional expression (Figure 1B and 1C). An adenovirus carrying the sequence of the Green Fluorescent Protein (Ad_GFP) at an MOI of 5 served as a control (see supplemental materials, available online at http://circres.ahajournals.org). No differences in cell appearance, uniformity of conduction, conduction velocity (CV), or APD were observed between Ad_GFP-infected and noninfected monolayers.
Single Cell Electrophysiology
Voltage clamp experiments were performed in single neonatal rat ventricular myocytes infected with Ad_IKs or Ad_GFP using a HEKA EPC 9/2 amplifier (HEKA Elektronik). 5-second depolarizing steps were applied in 10 mV increments from a holding potential of −50 mV while superfusing the cells with (in mmol/L): NaCl 130, KCl 5.4, MgCl2 1.0, CaCl 1.0, HEPES 10, glucose 10, nifedipine (20 μmol/L); pH=7.4 (NaOH). The pipette filling solution contained (in mmol/L): KCl 30, potassium aspartate 90, K2ATP 5.0, Hepes 5.0, EGTA 10, MgCl2 1.0, NaCl 15; pH=7.2 (KOH).
Current clamp experiments were conducted in HEK293 cells (ATCC, CRL1573) using an Axopatch 1D amplifier (Axon Instruments). HEK293 cells were transfected (Effectene Transfection Kit, Qiagen) with guinea pig cDNA of Kir2.1 subcloned in a pcEP4 vector and cotransfected with the human cDNA of minK-KvLQT1 subcloned in a pcDNA3 vector. An S1-S2 pacing protocol was applied in the presence of Tyrode solution, in which each 300-ms S1 stimulus was followed by a 50-ms S2 stimulus of equal magnitude at progressively shorter intervals. The pipette solution consisted of (in mmol/L): KCl 20, K-aspartate 90, KH2PO4 10, EDTA 5.0, K2ATP 1.9, HEPES 5.0, and Mg2+ 7.9; pH 7.2 (KOH).
All patch clamping experiments were performed at room temperature. Pipette DC resistances for both experiments ranged between 4 and 5MΩ.
Culture dishes were placed on a heating chamber and continuously superfused with HBSS without bicarbonate (Sigma) containing (in mmol/L): CaCl2 1.6, KCl 5.4, MgSO4 0.8, KH2PO4 0.4, NaHCO3 4.2, NaCl 136.9, NaHPO4 0.3, D-Glucose 5.5, and HEPES 10; pH 7.4 (NaOH). All experiments were performed at 35°C. Repetitive stimuli (duration, 5 ms; strength, twice diastolic threshold) were applied by a thin extracellular bipolar electrode at increasing frequencies, starting at 1 Hz, until loss of 1:1 capture or initiation of sustained reentry. Isoproterenol (200 nmol/L) was superfused when indicated. Electrical impulse propagation was recorded optically by staining the monolayers with the potentiometric dye di-8-ANEPPS (40 μmol/L; Molecular Probes) for 15 minutes. The changes in fluorescence corresponding to transmembrane voltage were recorded by an 80×80 pixel CCD camera (SciMeasure Analytical Systems Inc, Decatur, Ga) in 2-second movies at 500 frames per second (LabWindows Acquisition). Illumination was restricted to the time of recording to minimize the effects of phototoxicity limiting the exposure times to a range of 10 to 50 seconds. The signals were amplified, filtered, and digitized for off-line analysis. No electromechanical uncouplers were used for motion suppression. See the supplemental materials for further details.
Optical Data Analysis
For APD and CV measurements, average signals were obtained from individual optical movies recorded during repetitive pacing at varying frequencies. APD80 maps were constructed by measuring the time between the upstroke and 80% of repolarization in each pixel. For CV measurements, activation times were calculated for each pixel, and local conduction vectors were determined as described.16,17
Wavelength (WL) was determined during sustained reentry using the phase-mapping technique.18 WL was defined as the expanse between the wavefront and the end of the repolarization tail1 and measured at 5 mm from the center of rotation (see the supplemental materials for details). Sites of wavefront breakup (wavebreak) marked by phase singularities (PSs) were also identified in phase maps.19,20 PSs are defined as sites where all phases of the action potential converge.
Statistical analysis was performed with Origin software (version 7.0). One-way ANOVA was used for the analyses of CV, APD, WL, and frequency of reentrant activity. Regression analysis was performed on WL data. Values are expressed as mean±SE. A value of P<0.05 was considered significant.
Of a total of 85 monolayers, 44 were infected with “Ad_ IKs” (IKs channel adenovirus), and 41 were infected with “Ad_GFP” (GFP adenovirus). Altogether, 584 optical mapping movies were analyzed.
Spatially uniform protein expression was confirmed by immunohistochemistry in 10 multicellular preparations infected with Ad_IKs. Figure 1A shows an image of a sparsely plated neonatal rat ventricular myocyte preparation immunostained using a specific anti-KvLQT1 antibody (US Biological) in green. An average of 94±4% of the cells showed positive staining. Functional protein expression of the IKs channel was confirmed by whole cell voltage clamp. Figure 1B illustrates representative examples of response currents obtained from single neonatal rat ventricular myocytes infected with Ad_ IKs (left) and Ad_GFP (right) by applying the voltage clamp protocol depicted in the inset and described in the methods section. Although both groups of cells yielded similar peak outward currents in the first 20 ms of recordings, only the IKs-infected myocytes exhibited slowly activating pulse currents and slowly deactivating tail currents. Figure 1C shows the activation curve constructed from measurements at the end of the 5-second pulses (when the IKs-like currents are maximal). Under these steady-state conditions current densities in the IKs group were 5.2±1.4 times larger than in the control group. Finally, when compared with control, cells infected with Ad_IKs showed no apparent changes in intrinsic transmembrane currents, including ICa-L, Ito, and IK1. Further characterization of the infected myocytes can be found in the supplemental materials.
Action Potential Duration and Conduction Velocity
We then investigated the effects of IKs overexpression on APD in confluent electrically-coupled monolayers. Mean APD80 was determined from high-resolution APD maps during 1:1 activation in 32 control and 32 IKs monolayers. IKs decreased APD80 when compared with control but reached statistical significance only at the lower frequencies (1 Hz, P<0.0056; 2 Hz, P<0.0003; and 3 Hz, P<0.03) (see Figure 2). In addition, whereas in the control APD changed monotonically as a function of stimulation frequency, the IKs group had an initial bell-shaped profile demonstrating that IKs contributes more outward current to repolarization at 1 Hz (and at even slower frequencies) than at 3 Hz.21 Because IKs overexpression results in significant APD shortening only at relatively slow pacing frequencies in our monolayers, it is reasonable to hypothesize that the frequency of reentry in the IKs-overexpressing monolayers will be similar to the controls, unless the dynamics of reentry provide a substrate for further APD abbreviation. We therefore measured APD80 during reentry. As shown in Figure 3B, APD80 was shorter in IKs than control monolayers at all rotation frequencies (control slope: −7.31±0.89, IKs slope: −5.90±0.73; P=1.40×10−5).
During pacing, CV remained constant as the impulse traveled centrifugally from the stimulating electrode (see the supplemental materials). Under these conditions CV differences between control and IKs monolayers did not reach statistical significance. On the other hand, as expected,17,18,22 during reentry CV increased gradually when measured as a function of distance from the core in both groups as shown in Figure 3C and 3D. However, this increase was significantly less pronounced in the IKs monolayers, consistent with a less excitable preparation.17
IKs Overexpression Reduces Wavelength and Increases Rotor Frequency
The above results are in agreement with previous studies showing that, during sustained functional reentry, APD and CV are abbreviated in the vicinity of the center of rotation,1 which contributes to the significantly higher activation frequencies that are achievable during reentry compared with external pacing. We sought to address this issue by quantifying both WL and frequency during sustained reentry. Figure 3A shows snapshots of single spiral waves in control (left) and IKs-overexpressing (right) monolayers. Representative examples are available as movies in the supplemental materials. In Figure 3E we have plotted the WL measured for each rotor as a function of rotation frequency. Both groups showed similar frequency dependence; WL shortened in both as the rotation frequency increased (control slope: −0.81±0.36; IKs slope: −0.76±0.20). However, whereas control WLs ranged between 12.02 and 22.68 mm, IKs WLs were significantly shorter, ranging between 4.50 and 12.69 mm (P=2.87×10−9). Figure 4 compares the average rotation frequencies of 15 control monolayers with those of 20 littermate IKs monolayers. Individual rotor frequencies ranged between 4.9 and 13 Hz in both. On average, however, rotors in IKs monolayers were significantly faster (P<0.0436) than controls.
IKs is enhanced by β-adrenergic stimulation via cAMP/protein kinase A-dependent pathways.23 We therefore administered isoproterenol (200 nmol/L) to 10 monolayers infected with Ad_IKs and 10 control monolayers to assess its effect on both focal spontaneous activity, evident as typical target patterns with centrifugal propagation from the source, and self-sustained reentry (spiral waves). As shown in Figure 5, isoproterenol invariably increased the frequency of spontaneous focal discharges in both groups (A and B). However, it did not change the frequency of rotation during reentry in either group (C and D). A possible explanation for this finding could be that isoproterenol increases L-type calcium current which would tend to counteract the IKs activity.
Wavebreaks and Fibrillatory Conduction
Unlike control monolayers, the waves emanating from rotors in the IKs preparations often underwent wavebreak and formation of new, short-lived rotors in a time-dependent fashion. We will refer to this distinct pattern of electrical activity as fibrillatory conduction. As illustrated by the representative sequential phase maps presented in the top panel of Figure 6A, waves emanating from sustained rotors in control monolayers did not undergo wavebreak (0/15). In contrast, as illustrated in the bottom panel, stable rotors gave rise to multiple wavebreaks and fibrillatory conduction in 9 IKs preparations (see supplemental Movies). This corresponded to 45% of the IKs infected monolayers that sustained a stable rotor. The initial wavebreaks consistently occurred distally from the mother rotor and progressively “approached” it by impinging on incoming wavefronts resulting in more wavebreaks. The pattern eventually reached a steady state in which short-lived rotors coexisted with the main source, rotating at the same frequency. Figure 6B illustrates the patterns of evolution of wavebreaks with time. Each plot corresponds to a different monolayer and each point to an optical recording. Movies were sequential in time, as illustrated in panel A. In some instances the steady state fibrillatory conduction pattern was already advanced by the time of first recording. In the event that the mother rotor terminated, either another rotor took over as the source or all activity terminated. We did not find any episodes of fibrillatory conduction in which there was no driving source. As illustrated in Figure 7, both groups sustained rotors whose frequency overlapped over a range of 4.9 to 13 Hz. Yet only the IKs monolayers underwent wavebreaks and fibrillatory conduction. Significantly, wavebreaks occurred at the highest frequency values in the latter group.
Because β-adrenergic stimulation enhances IKs, we performed an additional set of experiments in 5 Ad_IKs- infected and 5 Ad_GFP-infected preparations to determine whether the exposure to 200 nmol/L isoproterenol would increase wavebreak density (as shown in Figure 6). We observed that the number of singularity points indeed increased in both groups but as a result of collisions with spontaneous foci rather than of wavebreaks (data not shown). This is likely to be attributable to isoproterenol enhancement of spontaneous activity as shown in Figure 5A and 5B.
To gain insight into possible mechanisms underlying IKs-induced wavebreaks and fibrillatory conduction, we used a heterologous expression system. Our objective was to determine the role of IKs on the threshold for excitation at diastolic potentials in the absence of other native cardiac-specific proteins that could confound the interpretation of the results. Using previously described methods,24 1 group of HEK293 cells was stably transfected with Kir2.1, responsible for IK1 to set the resting membrane potential at ≈−70 mV. A second group of HEK293 cells was cotransfected with Kir2.1 and KvLQT1-minK. Five cells in each group were subjected to a whole-cell current clamp S1-S2 stimulus protocol, as illustrated by the representative experiments in Figure 8. In Panel A, each square depolarizing pulse S1 (duration: 300 ms; frequency: 1–2 Hz) was followed by an S2 of equal magnitude, but briefer duration (50 ms), and the S1-S2 interval was varied, with the minimum S1-S2 duration adjusted so that S2 did not encroach on the S1 response. As shown in panel B, the amplitude and shape of the action potential-like response in cells expressing Kir2.1 alone was not affected by the timing of the S2 stimulus. In contrast, as shown in C, a clear time dependency was demonstrated in cells expressing both Kir2.1 and minK-KvLQT1 genes: progressively reducing the S1-S2 interval resulted in gradual changes in both the shape and amplitude of the S2 response, indicative of slow IKs deactivation.24 Similar results were obtained in all experiments surveyed in this manner. Taken together, these results support the idea that both IK1 and IKs are important in the recovery of the current requirements for excitation during the diastolic interval.
We have generated a 2-dimensional biological excitable medium to investigate the consequences of the overexpression of IKs on excitation, propagation, and the dynamics of reentrant activation. The most important results are: (1) In agreement with previous work, IKs overexpression shortens APD during pacing reaching statistical significance only at low stimulation frequencies (1–3 Hz); (2) During sustained reentry, IKs over-expression decreases APD, CV, and WL at frequencies ranging between 4.9 and 13 Hz; (3) Despite a significant WL shortening, IKs overexpression results in “history dependent” sink/source mismatch during diastole that leads to wavebreak and fibrillatory conduction particularly at high frequencies of reentry. Computer simulations presented in the supplemental materials show that a spatially heterogeneous distribution of such mismatch could account for the findings of increased wavebreaks in the experiments; (4) Current clamp experiments on HEK293 cells cotransfected with KCNJ2 and KCNQ1-KCNE1 show that the slow deactivation kinetics of IKs alone may be sufficient to induce wavebreak formation. These results, together with those of computer simulations and patch clamp data presented in the supplemental materials, allow the prediction that spatially distributed differences in IKs deactivation kinetics during diastole underlie the mechanism of IKs-induced wavebreaks and fibrillatory conduction demonstrated in our monolayers. Overall, the results provide the first demonstration that IKs involvement in postrepolarization refractoriness can lead to wavebreak formation and fibrillatory conduction.
Dynamics of Fibrillatory Conduction
Studies in hearts from several species ranging in size from the mouse to the pig18,22,25 have demonstrated that even a single rotor can result in ECG patterns that are indistinguishable from VF, which suggests that at least some forms of fibrillation are highly organized and depend on the uninterrupted periodic activity of a small number of high-frequency reentrant sources. The rapidly succeeding wavefronts emanating form such sources propagate throughout the ventricles and interact with tissue heterogeneities, both functional and anatomical, leading to fragmentation and wavelet formation,22,25 the end result being fibrillatory conduction.1,2 Apparently chaotic waves often recorded optically in intact hearts and attributed to fibrillatory conduction may be generated by sources outside the field of view. In our monolayers, on the other hand, the area recorded corresponds to the entire preparation, allowing the strong conclusion that all the episodes of fibrillatory conduction were sustained by at least 1 stable rotor. Occasionally, rotor activity was abolished by spontaneous focal activity, and this resulted in the immediate termination of fibrillatory conduction.
IKs Contribution to Repolarization
The contribution of IKs to action potential repolarization has been a matter of debate.26,27 Even in humans, both loss of function28 and gain of function29 of IKs can result in APD (QT) interval prolongation. Therefore, how the interplay between IKs and other currents modulates APD and, perhaps more importantly, its accommodation to rate, remains unresolved. In our experiments on neonatal rat ventricular monolayers we found that IKs significantly blunts the APD response to frequency (Figure 2). Nevertheless, at increased stimulation frequencies (into the range of rotor frequencies) the shortening of the APD in the IKs preparations did not reach statistical significance when compared on a frequency by frequency basis. During reentry, however, overall APDs were significantly shorter in IKs-overexpressing monolayers (Figure 3B). This may suggest that IKs enhances the repolarizing influence exerted by the core of the rotor to further abbreviate the action potential allowing for the slightly faster frequencies of rotation observed in Figure 4. Our data are not conclusive in this respect, and more studies beyond the scope of this one would be required to elucidate the exact role of IKs on cardiac repolarization.
Recovery of excitability after an action potential can be a slow process that greatly outlasts the action potential duration (APD).3 Previous studies in isolated guinea pig ventricular myocytes have shown that activation failure can occur at diastolic potentials (even if the Na+ current, INa, has had sufficient time to recover completely from inactivation) because of the slow gating kinetics of IK.4 This phenomenon is called postrepolarization refractoriness. We surmised that IKs, the slow component of IK, is an important determinant of postrepolarization refractoriness. Our experiments in HEK293 cells provide strong support to such an idea by revealing that IKs expression alone suffices to explain time dependent activation failure.
However, it was uncertain whether the results observed at the single cell level could be extrapolated to allow inferences related to the propagation of the electrical impulse where, in addition to cellular excitability, one has to consider electrical coupling and wavefront curvature,30 as well as structural heterogeneities, as potential contributors to the success or failure of propagation of the action potential. We found that IKs significantly shortens the wavelength while slightly but significantly increasing the frequency of reentry in the monolayers. Yet in this model, waves emanating from a stable source more readily encounter tissue that is repolarized but not yet excitable, leading to wave fragmentation. Such an apparent paradox can be readily explained by the results in the HEK293 cells showing that in the presence of full repolarization, the slow deactivation kinetics of IKs significantly impair the recovery of excitability at early diastolic intervals. This is not to say that the INa threshold, or the rate of recovery of INa and ICa, are not critical for cell excitability; however, under conditions in which these parameters are fully recovered at any given location, an increase in the number of IKs channels that remain open during diastole will act to oppose any depolarizing current. As such, the approach to threshold would be delayed with the possible occurrence of wavebreak.
Computer simulations (see supplemental materials) aided us in establishing that a spatial dispersion in the conformational state of the IKs channel, or spatially inhomogeneous recovery kinetics during diastole, may be sufficient to allow for wavebreak/reentry formation at specific locations. Therefore, the simplified nature of the monolayer, together with the results of simulations, allows us to postulate that the spatially distributed wavebreaks and intermittent block processes that are frequently observed during VF result in part from inherent spatial heterogeneities in IKs distribution and of its slow gating kinetics.
Role of IKs Accumulation
IKs activates in response to depolarization at potentials greater than −30 mV and reaches half-maximal activation at +20 mV. IKs activates more slowly than any other known K+ current and requires membrane depolarization in the order of seconds to reach a steady state,5 conditions that are never achieved in vivo. Although IKs activates slowly, because of its slow deactivation kinetics it will tend to accumulate at higher frequencies and therefore play an important role in VT/VF.31 Regardless of whether IKs accumulation occurs because of slow deactivation, β- adrenergic stimulation, rate-dependent conformational states, or all of the above, it is reasonable to expect that an increase in the magnitude of IKs during the action potential will have an effect on excitability. Provided that the previous history of depolarizations leaves a significant portion of repolarizing channels open, larger quantities of current will be needed for excitation immediately after the action potential has ended.
Excessive APD prolongation secondary to IKs reduction leads to early afterdepolarization (EAD)-induced arrhythmias.28,29 On the other hand, our experiments show that increased IKs can lead to reentry by means of wavebreak and singularity point formation. In fact, this could well be the mechanism underlying arrhythmias in some short QT syndrome patients.10,11
Most known class-III drugs block IKr and prolong APD.32 Unfortunately, IKr blockade typically causes excessive APD prolongation at slow heart rates. Blockade is much less effective at high rates.33 This so-called “reverse use-dependent action” can be pro-arrhythmic.32–34 Hence, selective IKs blockade may be an appropriate alternative with less pro-arrhythmic potential.
It is generally accepted that the role of IKs on excitability is species-dependent. Our neonatal rat ventricular myocyte model allowed us to express the human IKs channel and study its effects on conduction during pacing, sustained reentry, and fibrillatory conduction in a highly controlled environment. However it should be kept in mind that the channel is expressed in rodent myocytes with an ionic profile different from that of human myocytes. The presence of myofibroblasts in a monolayer is thought to be responsible for the low values of CV.35 Although 2-hour differential preplating and bromodeoxyuridine was used to reduce fibroblasts, total elimination cannot be achieved.
The conclusions that can be drawn from these results are conceptual and should be furthered in translational experiments before attempting to extrapolate them to the clinical setting.
We thank Li Gao, Jenny Deng, Michelle Auerbach, Helene Oberer, and Lisa Krapf for their expert technical support.
Sources of Funding
This work was supported by NHLBI Grants PO1 HL039707, RO1 HL070074, RO1 HL060843 (to J.J), AHA Postdoctoral Fellowship (to S.V.P), and the Swiss National Science Foundation (to A.G.K and S.R).
Original received February 1, 2007; revision received May 29, 2007; accepted June 27, 2007.
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