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1Bioelectricity Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston 02115; 2Cardiovascular Engineering Incorporated, Holliston, Massachusetts 01746; and 3Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Submitted 18 November 2002 ; accepted in final form 26 February 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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adenovirus; action potentials; early afterdepolarizations
We (13, 15) have shown that overexpression of a truncated Kv1 polypeptide (Kv1N206Tag) in the hearts of Kv1DN mice prolonged the action potential (AP) duration (APD), lengthened the QT interval, and increased the frequency of spontaneous and inducible polymorphic VTs. Electrophysiological studies of Kv1DN myocytes showed the elimination of a 4-aminopyridine (4-AP)-sensitive component of the slow inactivating outward K+ current (IK,slow) encoded by Kv1.5 (26). Ex vivo optical studies revealed prolonged APD with an apex-to-base dispersion of repolarization that was twofold greater than that in control mice. Consequently, programmed stimulation from the apex elicited reentrant circuits as the single premature beat captured, propagated toward the base, and encountered a functional line of block to initiate VT (1). Analysis of the cellular mechanism of the dominant negative effect of Kv1N206Tag demonstrated that the transgene trapped both Kv1.4 and Kv1.5 polypeptides in the endoplasmic reticulum (6), a mechanism similar to the one described for mutations in the carboxy terminus of HERG (5).
Previous studies have shown efficient adenovirus-mediated gene transfer into the hearts of several species, including mice (7–12, 18). On the basis of these studies, we hypothesized that the regional overexpression of Kv1.5 would increase the number of functional Kv1.5 channels reaching the plasma membrane of cardiocytes at the base of the heart, and this, in turn, would shorten the APD and QT intervals. We further hypothesized that shortening of the APD at the base of the left ventricle (LV) might be sufficient to abolish reentrant arrhythmias. Here, we show that targeted regional infection with adenoviral vectors encoding Kv1.5 (AV-Kv1.5) produces significant shortening of the APD and QT interval by augmenting the expression of 4-AP-sensitive, voltage-gated outward K+ currents.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-globin IVS III and a polyA signal. Similar adenoviral vectors encoding
the nuclear-localizing -galactosidase gene (AV-LacZ) and green
fluorescent protein (AV-GFP) were used. Virus was titrated to plaque-forming
units (pfu) per milliliter using plaque assays; equal numbers of viral
particles (5 x 108 pfu) in a sterile vehicle (3%
glycerol-0.9% saline) were used for all injections. Cell culture. HEK293 cells were grown in DMEM supplemented with 5% fetal calf serum (GIBCO-BRL) and penicillin-streptomycin in an incubator at 37°C with 5% CO2. Before infection, the culture medium was replaced with serum-free medium. Cultures were infected at multitudes of infection of 10 and 100 with AV-GFP alone or in combination with AV-Kv1.5. At 24 h postinfection, the cells were harvested and used either for Western blot analyses or patch-clamp experiments.
Surgical approach. All animal studies were performed in accordance with the guidelines of the Harvard Medical Area Standing Committee on Animals after approval by the Institutional Animal Care and Use Committee. All experiments were performed using heterozygous Kv1DN mice (15) (age: 3–10 mo), which were anesthetized with ketamine (50 mg/kg) and xylazine (50 mg/kg), intubated with a 22-gauge catheter, and ventilated with a Harvard Rodent Ventilator. Enflurane (Ethrane, Baxter) was used for maintenance of anesthesia. A midline skin incision was performed, and the chest and the pericardium were opened in the third intercostal space in the left midclavicular line. We performed a single intramyocardial injection of 25 µl of either vehicle or adenoviral vectors under a x10 magnifying operating microscope. The injection was targeted to the basal part of the LV free wall, and its success was immediately assessed by noting a brief paling and swelling of the ventricular wall. After the injections, the chest wall and skin were closed. The mice were extubated at the start of spontaneous respiration and placed on warmed bedding. All operations were performed under a heating lamp to maintain normal body temperature, and the ECG was monitored continuously.
Before this study, we established the surgical technique by intramyocardial injection of 25 µl of AV-LacZ (5 x 108 pfu) and assessment of the efficacy by X-Gal staining 7 days postinfection. Histological analysis showed that with optimal injection technique, most of the basal part of the LV free wall expressed LacZ, whereas the septum and right ventricle did not.
Western blots. For Western blot analyses, whole cell lysates of ventricles from control and infected Kv1DN mice were prepared by extraction with RIPA buffer. Lysates of HEK293 cells were solubilized in 1% Triton X-100. The blots were reacted with anti-FLAG M2 (Sigma) and visualized with a chemiluminescence detection system (Supersignal, Pierce).
Cellular electrophysiology and data analysis. Cardiac myocytes were obtained between 3 and 10 days after infection with adenoviral vectors or control injections using a modified Langendorff technique described previously (26). To identify successfully infected myocytes, AV-GFP (as a control) infection or AV-GFP/AV-Kv1.5 coinfections were performed (11). Whole cell voltage- and current-clamp recordings were conducted at room temperature (23°C). Experiments were performed using an Axopatch 200B patch-clamp amplifier interfaced with a DigiData 1322A analog-to-digital converter controlled by pCLAMP 8.1 software (Axon Instruments). For voltage-clamp experiments, the bath solution contained (in mM) 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose (pH 7.3 with NaOH). L-type Ca2+ and TTX-sensitive Na+ channels were blocked by the application of 2 mM CoCl2 and 20–200 µM TTX, respectively. For current- and voltage-clamp experiments, the pipette solution contained (in mM) 140 KCl, 1 MgCl2, 0.5 CaCl2, 10 HEPES, 5 EGTA, 5 ATP, and 0.5 GTP (pH 7.2 with KOH).
The patch electrodes were fabricated from 1.5-mm-diameter borosilicate
glass capillaries (WPI). Pipette resistances were 1–3 M
when
filled with the intracellular solution. In this study, the cell capacitances
in AV-GFP-infected and AV-Kv1.5-infected Kv1DN myocytes were 179.7 ±
9.3 pF (n = 17) and 185.5 ± 15.6 pF (n = 9,
P > 0.05), respectively. The series resistances were in the range
of 2–10 M and were compensated electronically by 75%. Voltage
errors resulting from the uncompensated series resistance were always <5 mV
and were not corrected. K+ currents were evoked by 4-s depolarizing
voltage steps to test potentials between -60 and +60 mV from a holding
potential of -70 mV after 20-ms prepulse to -20 mV to eliminate the residual
(TTX insensitive) component of the voltage-gated Na+ channels. Raw
traces are shown without correction for the leak currents (which were always
<100 pA). The 4-AP-sensitive current was obtained by subtraction of the
currents in the presence of 50 µM 4-AP from the currents under control
conditions. Both the resting membrane potential (RMP) and APs were recorded
using standard physiological solutions in the current-clamp mode. The APs were
evoked from the RMP by injections of a 500-pA current for 5 ms at 1 Hz. The
time constants of inactivation were determined using single- or
double-exponential fit by the Chebyshev method (Clamp-fit 8.1). Data analysis
and curve fitting were performed using Excel 2000 (Microsoft) and Origin 6.0
(Microcal; Amherst, MA).
The amplitude of endogenous currents in uninfected HEK293 cells at +60 mV was <200 pA. In most HEK-293 cells coinfected with AV-GFP and AV-Kv1.5, a typical outward K+ current was recorded. Current-voltage relationship was obtained using constant 250-ms pulses to potentials between -50 and +60 mV (in 10-mV increments) from a holding potential of -40 mV.
Infection of cardiomyocytes with AV-GFP alone did not result in the
expression of 4-AP-sensitive currents. When we studied cardiocytes infected
with AV-GFP and AV-Kv1.5, we detected 4-AP-sensitive currents in 17 of 24
green cells studied. We therefore concluded that the seven cells that did not
express the 4-AP-sensitive current were infected with GFP alone. Thus the
coinfection rate with both AV-GFP and AV-Kv1.5 was 70.8%. Because the hearts
were injected with a 1:1 ratio of AV-Kv1.5 and AV-GFP, the probability of
infection with each virus near the injection site is 
84.1% (the square
root of 70.8). Our analyses of coinfected cells represent green cells with
detectable 4-AP-sensitive currents.
Surface ECGs and in vivo electrophysiology. Before and 7 days after the operation, a 10-lead ECG (leads I-III, aVR, aVL, aVF, V1, V2, V4, and V6) was recorded for 1 min in anesthetized (ketamine-xylazine), spontaneously breathing animals (13). For the QT measurements, we averaged the 10 leads. As the previously published formula for QT correction applies only to physiological RR intervals for nonsedated animals (17), we established the RR/QT relationship using linear and nonlinear regression preoperatively in anesthetized animals. The predicted QT interval (QTpredicted) was calculated using the formula derived from the regression analysis, and the index QT interval (QTindex) was defined as QTobserved/QTpredicted. The QTindex was then retested by regression to confirm independence from RR intervals with the postoperative QT/RR data.
One week after the operation, animals underwent a transvenous, right ventricular electrophysiological study as previously described (3, 13). In vivo electrophysiological studies and ECG recordings were performed in four groups of Kv1DN animals injected with 1) 25 µl of 5 x 108 pfu AV-Kv1.5, 2) AV-GFP, 3) vehicle, or 4) sham-operated animals. All studies were performed and analyzed by a blinded operator.
Optical mapping of APs. Kv1DN mice were injected with either AV-LacZ (n = 10) or Av-Kv1.5 (n = 9). All optical measurements were performed, analyzed, and matched to the codes to obtain a blind study of mean APDs at 75% repolarization (APD75) ± SD and dispersion of repolarization. Hearts were stained with a voltage-sensitive dye (di-4-ANEPPS) (1). The activation time point at each site was determined from the maximum derivatives of the local AP upstroke (19, 20). Isochronal maps of activation, repolarization, and APD were generated as previously described (19).
Statistical analysis. For normally distributed values, we used Student's t-test (paired and unpaired) to compare the means of two groups and ANOVA with Bonferroni's post hoc test or Kruskal-Wallis test with Dunn's post hoc test for comparisons in more than two groups, where appropriate. Fisher's exact test was used for categorical variables. Analysis was performed with Prism 2.01 for Windows (Graphpad; San Diego, CA) and SPSS 10.0.1 (SPSS; Chicago, IL). All data are presented as means ± SE; n indicates the number of experiments, and a P value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Properties of AV-Kv1.5-infected Kv1DN myocytes. We next tested
whether AV-Kv1.5 codes for similar polypeptides when injected into the
myocardium. Western blot analysis using anti-FLAG antibody detected a
polypeptide with an apparent mobility of 76 kDa only in AV-Kv1.5-infected
Kv1DN mouse hearts (Fig.
1D). The localized injection with adenoviral vectors
caused only minimal damage at the site of the needle tract
(Fig. 1E). Injection
of AV-LacZ demonstrated a high level of expression around the injection site
at the base of the LV free wall (Fig.
1F), whereas the atria were not affected (data not
shown). The -galactosidase expression showed cell-to-cell variability;
in some cells, the staining was exclusively nuclear, whereas other cells
showed significant cytoplasmic staining.
We next compared outward currents in AV-GFP/AV-Kv1.5 (AV-Kv1.5)-coinfected Kv1DN myocytes with those in cells infected only with AV-GFP (control). Cells showing green fluorescence in both groups were investigated. The waveforms of depolarization-activated currents were distinct (Fig. 2, A–C). In AV-Kv1.5-infected cardiomyocytes (Fig. 2, B and C), peak outward and steady-state currents were higher than in AV-GFP-infected cardiomyocytes (Fig. 2A). The current-voltage relationship of the peak current amplitude (Fig. 2D) was linear, with mean values of 12.6 ± 1.2 pA/pF (n = 9) at +60 mV in AV-GFP-infected cardiomyocytes and 24.9 ± 3 pA/pF (n = 17, P < 0.05) in cells coinfected with AV-Kv1.5. The mean steady-state currents remaining after 4-s depolarization at +60 mV were 5.4 ± 0.6 and 11.2 ± 2.4 pA/pF, respectively (n = 17, P < 0.05; Fig. 2E). Thus the infection of AV-Kv1.5 resulted in a twofold increase in the peak and steady-state outward current densities.
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The time course of the outward current inactivation was fitted by a sum of
exponentials. With 4-s depolarizing pulses, two exponential components could
be resolved for all cells with the exception of those shown in
Fig. 2C. This analysis
revealed two time constants: a fast time constant (
fast) of
83.3 ± 14.3 ms and a slow time constant (slow)of 2.1
± 0.5 s for control (Fig.
2F,a; n = 10) and a fast of 55.5
± 4.6 ms and a slow of 2.5 ± 0.2 s for
AV-Kv1.5-infected cells (Fig.
2F,b; n = 6). The rapidly inactivating component
reflected the transient outward K+ current
(Ito) and was present in both AV-Kv1.5-infected and
control cells, and its amplitude remained unchanged (8.5 ± 1.2 vs. 5.8
± 1.5 pA/pF, P > 0.05). Inactivation kinetics of
Ito in both groups appeared independent of voltage. In
cells that exhibited markedly enhanced outward currents
(Fig. 2C), the
inactivation was best fitted by a single-exponential function with a
slow of 3.4 ± 0.6 s
(Fig. 2F,c; n
= 6). These cells either lacked the rapidly inactivating
Ito or had a small Ito that was masked
by the excessive expression of Kv1.5-encoded current.
Consistent with previous reports, infection of cardiomyocytes with AV-GFP alone did not show 4-AP-sensitive currents (data not shown). However, infection with AV-Kv1.5 resulted in the reappearance of 4-AP-sensitive currents in Kv1DN cardiomyocytes. Figure 3 shows a family of outward currents recorded under control conditions (A) and in the presence of 4-AP (B). The application of 50 µM 4-AP, which selectively blocks the 4-AP-sensitive component of IK,slow in wild-type mice, resulted in a marked reduction of peak and steady-state currents from 24.9 ± 3 and 11.2 ± 2.4 pA/pF (n = 17) to 17.9 ± 1.6 and 7.5 ± 1 pA/pF (n = 15), respectively. The subtracted 4-AP-sensitive current traces (Fig. 3C) and the current-voltage relationship (Fig. 3D) for the peak (open triangles) and steady-state currents (closed triangles) are shown. The density of the peak 4-AP-sensitive currents ranged from 7.8% to 73.2% of the total outward current, with a mean value of 25.9 ± 4.4% (n = 15) at +60 mV. The steady-state current density ranged from 0.2% to 22.8%, with a mean value of 6.3 ± 1.5% (n = 15). The inactivation decay of the 4-AP-sensitive current at +60 mV was best fitted by a single-exponential function (5.2 ± 1.5 s, n = 11). The presence of 4-AP-sensitive current as a component of steady-state currents is probably due to slow kinetics of inactivation.
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We next examined AP properties in single cardiomyocytes. Figure 4A illustrates superimposed APs recorded from control (AV-GFP alone) and AV-Kv1.5-infected cells. APDs were significantly shorter in AV-Kv1.5-infected myocytes (Fig. 4B); APDs at 50% and 75% repolarization were decreased from 17.1 ± 2.5 and 69.5 ± 8.2 ms in the control to 6.4 ± 1.8 and 16.9 ± 2.4 ms in AV-Kv1.5-infected cells (n = 11, P < 0.001), respectively. The RMP of the examined AV-Kv1.5-infected myocytes was not different from that of control cells (-67.6 ± 2.5 vs. -65 ± 3.3 mV, n = 11, P > 0.05). Similarly, AP amplitudes did not differ between AV-Kv1.5-infected and noninfected myocytes (108.9 ± 4.7 vs. 102.3 ± 5.7 mV, n = 11, P > 0.05). Finally, in 8 of 16 control cardiomyocytes, we observed EADs, as represented in Fig. 4C. In AV-Kv1.5-infected cells, the shortening of the APD was associated with the elimination of EADs in all myocytes studied (n = 21, P < 0.001).
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Optical maps of APs. Hearts from surgically treated mice often had pericardial scarring, fusion of the pericardium to the epicardium, and some tissue injury, which prevented optical recordings. Of the control (n = 10) and gene-rescued (n = 9) Kv1DN mice, only 5 of 10 AV-LacZ and 5 of 9 AV-Kv1.5 animals could be studied optically. Ventricular APD75 of AV-Kv1.5-infected mice had a mean of 32.8 ± 2.3 ms at the base and 23.9 ± 3.3 ms at the apex region (n = 5). Control or AV-LacZ-infected mice had a significantly longer APD75 (37.1 ± 3.7 ms) at the base (P < 0.05) and similar APD75 (25.1 ± 2.6 ms) at the apex (n = 5). Figure 5 illustrates AP recordings from an AV-Kv1.5-infected heart. At the injection site (1), APD75 were shorter compared with other sites at the base located 2–3 mm away from the injection site. AP recordings (traces 1–3) from the injection site (trace 1), another site at the base (trace 2), and from the apex (trace 3) show the different durations obtained from the epicardium.
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Surface ECGs and in vivo electrophysiology. We next compared the surface ECGs recorded on the seventh day after surgery with the preoperative ECGs (Fig. 6A). The preoperative mean RR and QT intervals did not differ between groups. Regression analysis of preoperative QT/RR data revealed a linear QT/RR relationship (R2 = 0.914, QTpredicted = 26.353 + 0.612RR) (Fig. 6B). Subsequent linear regression analysis demonstrated that the derived QTindex was independent of RR (R2 = 0.01). Postoperative RR and QT intervals were significantly shorter in the AV-Kv1.5-treated group (Table 1 and Fig. 6, C and D), whereas there were no significant differences among the other groups. Thus the AV-Kv1.5-induced shortening of the QT interval resulted in a higher heart rate. The effect was specific to this group and was not induced by either vehicle or AV-GFP. It is therefore unlikely that the surgical procedure or the viral infection itself caused this effect. Analysis of QTindex demonstrated a small incremental heart rate-independent postoperative shortening of the QT interval in AV-Kv1.5-infected animals (Fig. 6E; P < 0.05). Because the three control groups (sham, vehicle, and AV-GFP) were homogeneous regarding postoperative RR and QT intervals (P > 0.05), these groups were pooled to increase statistical power. An analysis of the postoperative QTindex of these groups versus the AV-Kv1.5 animals confirmed that the QTindex was shorter in the AV-Kv1.5 animals (Fig. 6E; P < 0.01).
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We next assessed the effects of regional heterogeneity in the change in QTindex by using a linear model with the 10 leads as a repeated measure and treatment as a grouping variable (Fig. 6F). In this model, the main effects of lead (P = 0.065) and the lead-by-treatment interaction (P = 0.104) were not significant, whereas the main effects of treatment remained highly significant (P = 0.002; Fig. 6F). The changes in QTindex in treated versus control animals were relatively small in the septal leads (V1 and V2) and were more clearly apparent in leads that are either adjacent to the terminal region of repolarization (AVL and V4–V6) or that have a significant vertical component (II and III), which allows them to detect the repolarization wave as it travels toward the base.
In vivo electrophysiological studies were successfully performed in a total of 43 Kv1DN mice (10 AV-Kv1.5 and 11 each in the AV-GFP, vehicle, and sham groups). Mean ventricular threshold values with 1.0-ms pulses were 0.38 ± 0.01 V (P > 0.05 between groups). Right ventricular effective refractory periods did not differ significantly between groups: sham, 66.4 ± 2.9 ms; vehicle, 65.3 ± 4.3 ms; AV-GFP, 66.6 ± 3.0 ms; and AV-Kv1.5, 61.8 ± 4.0 ms. VTs lasting greater than five beats were not inducible in any of the animals before isoproterenol treatment. Intraperitoneal isoproterenol raised the heart rate from 241.2 ± 10.8 to 387.9 ± 10.3 beats/min (P < 0.001), with no significant differences between groups. Only one VT lasting greater than five beats was elicited in a sham-operated animal after isoproterenol treatment. No sustained VTs were induced in any of the other groups (P > 0.05 between groups). Similarly, no differences were seen in the incidence of up to five extra beats after stimulation.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-subunit(s). Optical mapping studies confirm the effect of AV-Kv1.5 at the site of injection. Expression of Kv1.5 caused a significant shortening of optically measured APD75 in AV-Kv1.5-injected mice compared with AV-LacZ-injected mice. In addition, optical maps of the injection site demonstrated a regional shortening of APDs, as seen in Fig. 5, with no effect on distant sites. The shorter optical APD75 compared with the single cell data likely reflects pacing at a faster cycle length of 200 ms at 37°C. Moreover, in single cell analyses, there is no cell-cell coupling. In optical studies, the AP represents the sum of APs from hundreds of cells that are electrically coupled such that the cell with the shortest APD will drive the premature repolarization of adjacent cells with longer APDs.
Analysis of the ECGs revealed a shortening of the QT interval in mice injected with AV-Kv1.5 but no significant effect in any other group. Our preoperative ECG data demonstrate a tight linear correlation between RR and QT intervals at the slow heart rates during ketamine-xylazine anesthesia. This finding enabled us to derive a correction formula to compare QT intervals at these heart rates. The AV-Kv1.5-infected mice showed a significantly higher postoperative heart rate than the other groups. However, the shortening of the QT interval in the AV-Kv1.5 group persisted despite correction for the effects of the faster heart rates. It is likely that targeting of AV-Kv1.5 to the base of the LV, an area that is the last to repolarize (1), modified the end of the vectorial average of the repolarization wave and thus shortened the T wave and QT interval. We believe that the significant increase in the heart rate of the group with a genetically induced decrease in QT interval may represent compensation for the reduced systolic ejection time and stroke volume. These data suggest a reciprocal relationship between QT interval and heart rate such that a primary shortening of the QT interval leads to faster heart rates just as a primary increase in heart rate may shorten QT.
Transvenous programmed ventricular stimulation of the four groups showed that all mice were noninducible except a single sham-operated mouse. This contrasts with our previous studies, in which 40–50% of Kv1DN mice were inducible (3, 13). The low prevalence of VT inducibility in sham-operated animals suggests that the surgical procedure itself altered cardiac excitation and abolished the in vivo inducibility of VTs in these mice. We therefore could not determine whether the local rescue of the cellular phenotype and the shortening of the QT interval had an antiarrhythmic effect in vivo.
In summary, we demonstrated that in vivo infection of Kv1DN myocardium with AV-Kv1.5 reconstitutes the 4-AP-sensitive current (albeit with modified inactivation kinetics) and shortens the APD. In addition, shortening of the APD eliminated EADs and was associated with shortening of the QT interval and an increase in heart rate recorded in sedated animals. Thus targeted expression of functional channels may lead to new approaches for therapeutic interventions in patients with cardiac arrhythmias and LQTS.
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ACKNOWLEDGMENTS |
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G. Salama and G. Koren are recipients of grants from the National Heart, Lung, and Blood Institute.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* M. Brunner and S. A. Kodirov contributed equally to this study. 
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and 
, n = 9)
and Kv1.5-infected cells (
and 
, n = 17, P <
0.03), respectively. F: inactivation kinetics of the outward currents
(a–c). Current traces at 60 mV (points) and fitting curves
(solid line) were superimposed (r = 0.99 in all cases).
) and steady-state (
) 4-AP-sensitive
currents (n = 15). The scale bar is identical for all traces.
