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(Circulation. 2004;110:3435-3443.)
© 2004 American Heart Association, Inc.

Genetics

In Vivo Cardiac Gene Transfer of Kv4.3 Abrogates the Hypertrophic Response in Rats After Aortic Stenosis

Djamel Lebeche, PhD^*; Roger Kaprielian, PhD^*; Federica del Monte, MD, PhD; Gordon Tomaselli, MD; Judith K. Gwathmey, VMD, PhD; Arnold Schwartz, MD; Roger J. Hajjar, MD

From the Cardiovascular Research Center (D.L., R.K., F.d.M., R.J.H.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass; the Institute of Molecular Pharmacology and Biophysics (D.L., A.S.), University of Cincinnati Medical Center, Cincinnati, Ohio; the Johns Hopkins University School of Medicine (G.T.), Baltimore, Md; and Boston University School of Medicine (J.K.G.), Boston, Mass.

Correspondence to Roger J. Hajjar, MD, Cardiovascular Research Center, Massachusetts General Hospital, 149 13th St, CNY-4, Charlestown, MA 02129. E-mail hajjar{at}cvrc.mgh.harvard.edu

Received June 29, 2004; revision received August 10, 2004; accepted August 17, 2004.

	Abstract

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Background— Prolongation of the action potential duration (APD) and decreased transient outward K⁺ current (I_to) have been consistently observed in cardiac hypertrophy. The relation between electrical remodeling and cardiac hypertrophy in vivo is unknown.

Methods and Results— We studied rat hearts subjected to pressure overload by surgical ascending aortic stenosis (AS) and simultaneously infected these hearts with an adenovirus carrying either the Kv4.3 gene (Ad.Kv4.3) or the ß-galactosidase gene (Ad.ß-gal). I_to density was reduced and APD₅₀ was prolonged (P<0.05) in AS rats compared with sham rats. Kv4.2 and Kv4.3 expressions were decreased by 58% and 51%, respectively (P<0.05). AS rats infected with Ad.ß-gal developed cardiac hypertrophy compared with sham rats, as assessed by cellular capacitance and heart weight–body weight ratio. Associated with the development of cardiac hypertrophy, the expression of calcineurin and its downstream transcription factor nuclear factor of activated T cells (NFAT) c1 was persistently increased by 47% and 36%, respectively (P<0.05) in AS myocytes infected with Ad.ß-gal compared with sham myocytes. In vivo gene transfer of Kv4.3 in AS rats was shown to increase Kv4.3 expression, increase I_to density, and shorten APD₅₀ by 1.6-fold, 5.3-fold, and 3.6-fold, respectively (P<0.05). Furthermore, AS rats infected with Ad.Kv4.3 showed significant reductions in calcineurin and NFAT expression. (P<0.05).

Conclusions— Downregulation of I_to, APD prolongation, and cardiac hypertrophy occur early after AS, and in vivo gene transfer of Kv4.3 can restore these electrical parameters and abrogate the hypertrophic response via the calcineurin pathway.

Key Words: hypertrophy • gene therapy • ion channels • potassium • stenosis

	Introduction

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In response to excess hemodynamic loading and neurohormonal activation, the myocardium undergoes adaptive hypertrophic growth to temporarily augment cardiac function. Cardiac hypertrophy is associated with an increased cardiovascular morbidity and mortality,¹ which is in part due to electrical remodeling. A central and consistent electrophysiological change in cardiac hypertrophy is usually manifested by prolongation of the action potential duration (APD), which increases the propensity to develop lethal cardiac arrhythmias.² At the cellular level, APD prolongation is attributed to a reduction of a hyperpolarizing current provided by the calcium-independent transient outward K⁺ channel (I_to),^3–6 with concomitant reductions in Kv4.2 and Kv4.3 expression.^5,7,8 A reduction in I_to density and APD prolongation represent early electrical remodeling events in the diseased myocardium,⁹ pointing toward a potential role in disease initiation and progression.

Although considerable progress has been made in understanding the molecular pathogenesis of hypertrophy, the role of electrical remodeling associated with hypertrophy remains limited. Furthermore, the relation between I_to density, APD prolongation, and cardiac hypertrophy in vivo is unknown. Reduction of I_to density and prolongation of the APD can regulate calcium entry via the voltage-sensitive calcium channels and augment calcium transient ([Ca²⁺]_i) amplitude in cardiac myocytes,^4,10,11 which may help to support contraction of the compromised myocardium but may also harm the myocardium by increasing the propensity to develop arrhythmias^3,12 and by activating hypertrophic signaling pathways. Activation of the calcineurin pathway and onset of cardiac hypertrophy have also been reported in neonatal rat ventricular myocytes with reduced I_to by dominant-negative inhibition and pharmacological blockade of Kv4.2-based I_to channels. In addition, abnormal calcium handling in heart failure seems to be correlated with calcineurin activation.¹³ In the present study, we wanted to determine whether in vivo adenoviral gene transfer of Kv4.3-based I_to channels can increase I_to density, shorten the APD, and modulate the signaling pathways that regulate the hypertrophic response.

	Methods

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Adenovirus Preparation
Recombinant adenoviruses were generated with the E1-deleted pAdEasy-1 adenoviral plasmid (kindly provided by Dr Bert Vogelstein, John Hopkins University, Baltimore, Md) and the shuttle vector pAdTrack containing the cDNA for enhanced green fluorescent protein (GFP).¹⁴ An adenoviral vector encoding Kv4.3 (Ad.Kv4.3) was constructed by using the human Kv4.3 homologue (hKv4.3.1 cDNA-short splice variant).¹⁵ The Ad.Kv4.3 contained 4 copies of cardiac tissue–specific promoter, namely, the 250-bp fragment of the myosin light chain-2v gene, which we have shown to confer cardiac expression specificity. Ad.ß-gal contained cytomegalovirus–driven expression cassettes for ß-galactosidase and GFP.

In Vivo Adenoviral Gene Transfer and Induction of Ascending Aortic Stenosis (AS)
The method of adenoviral delivery was previously described by our group.^16,17 After baseline echocardiographic measurements, animals were randomly assigned to sham, control, and experiment groups. The method of adenoviral in vivo delivery was performed as previously described. In brief, male Sprague-Dawley rats (body weight, 250 to 300 g) were anesthetized with ketamine HCl (60 mg/kg IP) and xylazine (10 mg/kg IP) and placed on a ventilator. A 22-gauge catheter containing 300 µL of a saline solution or an adenovirus solution containing 1.5x10¹⁰ plaque-forming units of either AdLacZ or Ad.Kv4.3 was advanced from the apex of the left ventricle to the aortic root. A tourniquet was placed around the aorta and the pulmonary artery at a site distal to the tip of the catheter, and the solution was injected. The tourniquet was held in place for 40 seconds while the heart pumped against a closed system (isovolumetrically) and then released. The rats were then subjected to supravalvular aortic banding with a 0.58-mm (internal diameter) tantalum clip as previously described.¹⁷ Sham-operated animals were used as controls.

Echocardiography
Animals were lightly anesthetized with ketamine HCl (50 mg/kg IP) and xylazine (10 mg/kg IP), shaved, and placed in a left lateral position. Transthoracic 2-dimensional and M-mode echocardiographic images were obtained at the midpapillary level with a fully digitized Vingmed System Five (GE Vingmed Ultrasound) with a 10-MHz pediatric transducer. Images were recorded on a magneto-optical disk and subsequently analyzed with EchoPac software (version 6.0, GE Vingmed Ultrasound). Anterior and posterior wall thicknesses and left ventricular diameters in end-diastole (LVDD) and end-systole (LVDS) were measured offline from M-mode recordings, according to the American Society for Echocardiography leading-edge method. Fractional shortening (in percent) was calculated as [100x(LVDD–LVDS)/LVDd]. All measurements were averaged over 3 consecutive cycles and were performed by an investigator blinded to the experimental protocol.

Isolation of Ventricular Myocytes
On day 8, left ventricular myocytes were isolated for electrophysiological studies and protein analysis as previously described.⁴ Hereafter, myocytes derived from sham-operated rats will be referred to sham myocytes, whereas myocytes derived from rats subjected to aortic stenosis will be referred to as AS myocytes.

Single-Cell Electrophysiology
Current densities and action potentials (APs) were recorded by the whole-cell patch-clamp technique with an Axopatch 200B amplifier and a headstage CV 203 BU coupled to a Digidata 1320 personal computer interface (Axon Instruments). Cell capacitance was estimated by integrating the area of the capacitance transients after a 5-mV step from a holding potential of –70 mV. The measured currents were divided by the cell capacitance to normalize currents for cell size.

To measure I_to densities and APs, myocytes were superfused with a modified Tyrode’s solution containing (in mmol/L) NaCl 140, MgCl₂ 1, HEPES 10, CsCl 4, CaCl₂ 1, and D-glucose 10, with pH adjusted to 7.4 by addition of NaOH. CdCl₂ (0.3 mmol/L) was routinely added to block I_Ca,L when K⁺ currents were being recorded. Patch pipettes were pulled, fire-polished to final tip resistances of 2 to 4 m, and then filled with pipette solution containing (in mmol/L) potassium aspartate 130, KCl 20, HEPES 10, MgCl₂ 1, NaCl 5, EGTA 10, and MgATP 5, with pH adjusted to 7.2 by addition of Trizma base. Junction potentials (–8.2 mV) after rupture were corrected for AP recordings only. I_to was measured by depolarizing the myocyte membrane to +70 mV for 500 ms from a resting potential of –80 mV. Tip potentials were zeroed before formation of the membrane pipette seal in the Tyrode’s solution. Mean seal resistance averaged 12.5±0.9 G, and pipette resistance averaged 2.7±0.1 m. Voltage-clamp experiments were performed with an interepisode interval of 5 seconds. APs were initiated by short depolarizing current pulses (2 to 3 ms, 500 to 800 pA) at 0.2 Hz. In the case of AS myocytes, recordings were made only in GFP-positive cells. A xenon arc lamp was used to view GFP at 488/530 nm (excitation-emission), which confirmed adenoviral infection. All recordings were performed at room temperature (19°C to 23°C) within 18 hours of isolation on calcium-tolerant, quiescent, rod-shaped myocytes with clear and regular cross striations.

Calcium Handling and Contractility
Measurements of intracellular calcium and cell shortening were performed as described earlier. Myocardial cells were loaded with the Ca²⁺ indicator fura 2 by incubating the cells in medium containing 2 µmol/L fura 2-AM (Molecular Probes) for 30 minutes. Intracellular calcium concentration was calculated according to the formula [Ca²⁺]_i=K_d(R–R_min)/(R_max–R)B, where R is the ratio of fluorescence of the cell at 360 and 380 nm; R_max and R_min represent the ratios of fura 2 fluorescence in the presence of saturating amounts of calcium and effectively "zero calcium," respectively; K_d is the dissociation constant of Ca²⁺ from fura 2; and B is the ratio of fluorescence of fura 2 at 380 nm in zero Ca²⁺ and saturating amounts of Ca²⁺. The myocardial cells were imaged with a CCD video camera (Javelin Electronics) attached to the microscope, and motion along a selected rastor line segment was quantified by a video motion-detector system (Ionoptix).

Western Blot Analysis
Protein samples were prepared from sham and AS myocytes immediately after myocyte isolation. They were matched for protein concentration, separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), and transferred onto nitrocellulose membranes. Blots were incubated with antibodies against Kv4.2, Kv4.3, and Kv1.4 -subunits (Chemicon); calcineurin; nuclear factor of activated T cells (NFAT) 3; GFP (all from Santa Cruz); and dual phosphospecific antibodies to extracellular signal–regulated protein kinase 1 and 2 (ERK1/2), p38 (New England Bio Labs), and stress-activated protein kinase (SAPK, Promega) or total antibodies (Santa Cruz) overnight at 4°C. Membranes were incubated with appropriate secondary antibody conjugated to horseradish peroxidase for 2 hours, and bands were visualized by their chemiluminescence. Films from at least 3 independent experiments were scanned, and densities of the immunoreactive bands were evaluated with NIH Image software. The mitogen-activated protein kinase (MAPK) activities were verified by determination of the phosphorylation level of each kinase in myocardial lysates with the use of specific phosphoantibodies. Total protein contents of the corresponding MAPKs were also determined after stripping the phosphoblots to verify protein loading.

Statistical Analysis
All data are expressed as mean±SEM. Statistical comparisons between groups were made with 1-way ANOVA, followed by a Student-Newman-Keuls post hoc test, with the SPSS program (version 7.0 for Windows, SPSS Inc). Probability values <0.05 were deemed statistically significant.

	Results

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Adenoviral Gene Transfer In Vivo
Eight days after gene transfer, 20% to 30% of viable myocytes were shown to exhibit green fluorescence, as shown in Figure 1(A, direct light, and B, fluorescent). The green fluorescence of myocytes verified infection with Ad.ß-gal coexpressing GPF. GFP expression was also confirmed by Western blotting, as shown in Figure 1C. The fraction of infected cells was determined by counting green myocytes expressing GFP (infected cells) and nongreen myocytes (normal cells) in a given field by light and fluorescence microscopy. The following were the fractions recorded for the different groups: sham+Ad.ß-gal, 34±5% (n=6), and aortic banding+Ad.ß-gal, 29±4% (n=7). Echocardiography was performed before (baseline) and 7 days after viral injection and aortic banding as shown in the Table. The aortic-banded hearts transduced with Ad.ß-gal developed marked left ventricular hypertrophy, characterized by a 20% to 30% increase in the thickness of the posterior wall at end-diastole. Cross sections of harvested hearts were prepared, and dimensional measurements were carried out. The data demonstrated that a pronounced left ventricular hypertrophy developed in the hearts transduced with Ad.ß-gal and AS, with significant increases in left ventricular weight–body weight ratio, increases in interventricular septal thickness, increases in anterior wall thickness, and increases in posterior wall thickness. However, hypertrophy was not seen in aortic-banded hearts transduced with Ad.Kv4.3. Taken together, these data suggest that overexpression of Kv4.3 inhibits the development of pressure overload–induced cardiac hypertrophy. However, the data in the Table also show that there is a trend toward left ventricular dimensions in rat hearts with AS+Ad.Kv4.3. This can be explained by the fact that if the ventricle is blocked from developing hypertrophy under pressure-overload conditions, it will increase its left ventricular chamber size to generate the appropriate pressure to overcome the aortic banding. The heart utilizes the Starling curve to increase left ventricular pressure by operating at a higher point on the left ventricular volume axis. For that reason, it was not surprising to see a higher ventricular dimension in the AS+Ad.Kv4.3 hearts. It remains to be determined whether long-term infection with Ad.Kv4.3 and long-term inhibition of cardiac hypertrophy would significantly modify left ventricular dimensions.

View larger version (40K): [in this window] [in a new window]   Figure 1. Left ventricular myocytes derived from rat hearts after in vivo adenoviral gene transfer. Rod-shaped left ventricular myocytes isolated 8 days after catheter-based gene transfer of ß-gal gene coexpressing GFP with use of Ad.ß-gal. Four of 10 myocytes are shown expressing GFP and visualized under bright-field and fluorescent illumination (A) and fluorescent illumination alone at 510 nm with single excitation peak at 490 nm blue light (B). Infected myocytes are readily distinguishable from noninfected myocytes. C, Western blot of myocyte lysates shown in A or B, demonstrating GFP expression with increasing amounts of protein. Abbreviations are as defined in text.

View this table: [in this window] [in a new window]   Echocardiographic Parameters at Baseline and 7 Days After AS

Effect of Kv4.3 Overexpression on I_to Density and APD
I_to is frequently implicated in the electrical remodeling of cardiac hypertrophy and failure. Figure 2A shows raw traces of I_to recorded from sham and AS myocytes infected with Ad.ß-gal or Ad.Kv4.3 in response to depolarizing step voltages from –40 to +70 mV from a holding potential of –80 mV. The corresponding mean I_to densities, defined as peak current elicited by the depolarizing voltage step minus the steady-state current remaining at the end of a 500-ms voltage step, are presented in Figure 2B. I_to density was decreased in AS myocytes derived from hearts infected with Ad.ß-gal (9.9±1.0 pA/pF, n=18) compared with myocytes derived from sham hearts (17.6±0.80 pA/pF, n=27, P<0.05, at +40 mV). Overexpression of Kv4.3 resulted in high, robust I_to densities (52.3±7.0 pA/pF, n=12), which were significantly larger compared with that in AS myocytes infected with Ad.ß-gal or sham myocytes (Figure 2A and 2B).

View larger version (27K): [in this window] [in a new window]   Figure 2. In vivo expression of Ad.Kv4.3 increased Ito density in left ventricular myocytes subjected to AS. A, Representative whole-cell Ito currents recorded from –40 to +70 mV in sham-operated, Ad.ß-gal–infected, or Ad.Kv4.3-infected cells. Electrophysiological recordings were performed only on isolated myocytes displaying green fluorescence. B, Summary data of peak current-voltage relations of Ito. Ito current amplitudes were normalized to cell capacitance in each cell (pA/pF). As expected, Kv4.3 overexpression generated robust increase in Ito current and density. Shown are means (symbols) and SE (bars). *Ad.Kv4.3 vs Ad.ß-gal, P<0.05; sham vs Ad.Kv4.3, P<0.05. Abbreviations are as defined in text.

To investigate the molecular basis for the changes in I_to density, we examined the expression of Kv4.2, Kv4.3, and Kv1.4 -subunits, which represent candidate voltage-dependent K⁺ channels encoding I_to in the rat ventricle. Figure 3 shows typical Western blots demonstrating immunoreactive proteins for Kv4.3 (A), Kv4.2 (B), and Kv1.4 (C) in left ventricular myocytes derived from sham and AS hearts infected with Ad. ß-gal and Ad.Kv4.3. Consistent with a reduction in I_to density, the expression of Kv4.3 decreased by 50% in AS myocytes infected with Ad.ß-gal (77.8±4.2 AU, n=4) compared with sham myocytes (158.8±6.9 AU, n=4 P<0.05; Figure 3A). Similarly, the expression of Kv4.2 decreased by 58% in AS myocytes infected with Ad.ß-gal (46.3±9.6 AU, n=4) compared with sham myocytes (110.9±2.3 AU, n=4 P<0.05; Figure 3B). Unlike Kv4.3 and Kv4.2, Kv1.4 protein expression did not differ (6.4%) between AS myocytes infected with Ad.ß-gal (143.7±3.2, n=4), and sham myocytes (153.6±3.3, P>0.05) as shown in Figure 3C. Overexpression of Kv4.3 increased significantly in AS myocytes infected with Ad.Kv4.3 (123.4±7.5 AU, n=4) compared with AS myocytes infected with Ad.ß-gal (77.8±4.2 AU, n=4 P<0.05; Figure 3A) but had no effect on the expression of Kv4.2 (Figure 3B) and Kv1.4 (Figure 3C), which is consistent with our in vitro recordings (data not shown). These data demonstrate that the reduction in I_to density mirrors the reduction in Kv4.2 and Kv4.3 expression observed after AS and that in vivo adenoviral gene transfer of Kv4.3 can specifically increase the expression of Kv4.3-based I_to.

View larger version (44K): [in this window] [in a new window]   Figure 3. Effect of in vivo adenoviral gene transfer of Kv4.3 on candidate Kv -subunits known to encode Ito. Cell lysates were matched for protein concentration, separated by SDS-PAGE, and then transferred to nitrocellulose membranes. Blots were incubated with corresponding antibodies against Kv4.3 (A), Kv4.2 (B), and Kv1.4 (C), followed by detection with enhanced chemiluminescence. Data are expressed as mean±SEM. *Ad.Kv4.3 vs Ad.ß-gal, P<0.05; sham vs Ad.ß-gal, P<0.05; sham vs Ad.Kv4.3, P<0.05. Abbreviations are as defined in text.

Because I_to is an important determinant of early repolarization of rat cardiac APs, we anticipated that augmentation of I_to density would shorten the APD. Figure 4A shows typical APs recorded from sham and AS myocytes infected with Ad.ß-gal and Ad.Kv4.3, with Figure 4B summarizing averaged APD at 50% (APD₅₀) and 90% (APD₉₀) repolarization. AS caused a significant prolongation of both APD₅₀ (3.9±0.6 ms, n=27, versus 9.8±1.8 ms, n=10; P<0.05) as well as APD₉₀ (23.9±2.2 ms, n=27, versus 50.3±4.3 ms, n=10; P<0.05) compared with sham-operated controls. Expression of robust I_tos shortened both APD₅₀ (2.7±0.5 ms, n=10) and APD₉₀ (10.6±2.2 ms, n=10) in AS myocytes infected with Kv4.3 compared with AS myocytes infected with ß-gal. These data suggest that I_to is reduced and APD prolongation occurs 8 days after AS and that in vivo adenoviral gene transfer of Kv4.3 can increase I_to density and shorten APD over and above levels observed in sham-operated animals.

View larger version (28K): [in this window] [in a new window]   Figure 4. In vivo expression of Ad.Kv4.3 shortened APD in left ventricular myocytes subjected to AS. A, Representative AP traces from sham, Ad.ß-gal–infected, or Ad.Kv4.3-infected cells. Horizontal bars indicate 0 mV. B, Mean APD evaluated at APD50 and APD90 repolarization. Aortic banding caused pronounced prolongation of APD, which was significantly shortened to below sham levels by Kv4.3 overexpression. *Ad.Kv4.3 vs Ad.ß-gal, P<0.05, for APD50 and APD90; sham vs Ad.ß-gal, P<0.05; sham vs Ad.Kv4.3, P<0.05. Abbreviations are as defined in text.

The fast components of I_to fitted with monoexponentional functions were 47.7±4.7 ms (n=17), 55.0±2.3 ms (n=17, P=0.01, compared with sham), and 33.8±1.8 ms in sham, AS+Ad.ß-gal, and AS+Ad.Kv4.3 groups (n=7, P=0.01 compared with AS+Ad.ß-gal), respectively.

Effect of Kv4.3 Overexpression on Calcium Handling and Contractility
Central to our hypothesis was that the hypertrophic response was associated with increased intracellular calcium. Elevations in intracellular calcium as a result of APD prolongation may provide an important stimulus for the activation of stress pathways, including calcineurin and MAPKs, and induction of hypertrophy. Figure 5 shows recordings from representative cardiomyocytes isolated from sham and aortic-banded hearts infected with Ad.ß-gal or Ad.Kv4.3. AS was associated with increases in peak calcium and cell shortening when compared with sham (Figure 5A and 5B). Overexpression of Kv4.3 attenuated the enhanced contractile function seen with AS+Ad.ß-gal hearts (Figure 5A and 5B).

View larger version (31K): [in this window] [in a new window]   Figure 5. In vivo expression of Ad.Kv4.3 improves contraction and calcium handling in left ventricular myocytes subjected to AS. A, Representative recordings of myocyte cell shortening and Ca2+ kinetics from sham, AS+Ad.Kv4.3, and AS+Ad.ß-gal hearts. AS hearts showed increased shortening and prolonged Ca2+ transients compared with sham. Ad.Kv4.3 normalized these parameters. B, Tabulated parameters of Ca2+i and cell shortening in sham, AS+Ad.Kv4.3, and AS+Ad.ß-gal hearts; sham vs Ad.ß-gal, P<0.05; *Ad.Kv4.3 vs Ad.ß-gal, P<0.01. Abbreviations are as defined in text.

Effect of Kv4.3 Overexpression on Hypertrophy and Activation of MAPK Pathways
AS was associated with global and cellular hypertrophy on day 8 as shown in the Table. AS rats infected with Ad.ß-gal developed hypertrophy compared with sham-operated rats, as assessed by the significant increase in myocyte capacitance (119±4.3 pF, n=30, versus 189±14 pF, n=30; P<0.05) and heart weight–body weight ratio (0.30±0.03 mg/g, n=6, versus 0.44±0.03 mg/g, n=10; P<0.01). Kv4.3 overexpression was associated with an attenuation of cardiac hypertrophy, as observed by a reduction in myocyte capacitance (150±15 pF, n=28, P<0.05) and a reduction in heart weight–body weight ratio (0.34±0.04 mg/g, n=10; P<0.05). The specificity of the Kv4.3 effect was confirmed by measuring the capacitance in the AS+Ad.Kv4.3 group of GFP-negative myocytes, which had a capacitance comparable to AS+Ad.ß-gal GFP-negative cardiomyocytes (221±10 pF, n=33, versus 227±13 pF, n=32). The GFP-positive cardiomyocytes isolated from AS+Ad.Kv4.3 cells had a lower capacitance than that of the AS+Ad.ß-gal GFP-positive cardiomyocytes (150±15 pF, n=28, versus 221±10 pF, n=33; P<0.05).

We next sought to determine whether short-term AS could activate MAPK pathways, namely, the ERK1/2, SAPK, or c-Jun NH₂-terminal kinase (JNK) pathways and p38 and whether overexpression of Kv4.3-based I_to channels could modulate these pathways. The activity of ERK1/2 was increased by 1.97-fold in AS myocytes infected with Ad.ß-gal (0.61±0.04 AU, n=4) compared with sham myocytes (0.31±0.03 AU, n=4, P<0.05;Figure 6A).

View larger version (38K): [in this window] [in a new window]   Figure 6. Effect of in vivo adenoviral gene transfer of Kv4.3 on MAPK activity. Western blotting was performed on cell lysates from sham, Ad.ß-gal–infected, and Ad.Kv4.3-infected hearts with phosphospecific antibodies to ERK (A), SAPK (B), and p38 (C). Aortic banding induced activation of 3 MAPK pathways in parallel. Bar graphs are quantifications of MAPK activities. Activity of each MAPK was normalized against its corr esponding total protein (total ERKs=ERK1+ ERK2; total SAPK=p54SAPK+p46SAPK; total p38). Data, from 3 experiments, are presented as mean±SE. Sham versus Ad.ß-gal, P<0.05; sham vs Ad.Kv4.3, P<0.05. Abbreviations are as defined in text.

Similarly, the activity of SAPK/JNK was increased by 2-fold in AS myocytes infected with Ad.ß-gal (0.75±0.06 AU, n=4) compared with sham myocytes (0.37±0.02 AU, n=4 P<0.05; Figure 6B). The phosphorylation of p38 was also increased by 2.29-fold in AS myocytes infected with Ad.ß-gal (1.21±0.01 AU, n=4) compared with sham myocytes (0.53±0.01 AU, n=4 P<0.05; Figure 6C). The degree of activation of these kinases was similar to uninfected AS myocytes (data not shown), demonstrating that pressure overload leads to sustained phosphorylation of ERK1/2, SAPK/JNK, and p38 pathways. In vivo overexpression of Kv4.3 had no effect on phosphorylation of the 3 MAPKs after AS (Figure 6A–6C). Western blot analysis confirmed the equivalent expression of each of the total kinases in sham and AS myocytes infected with Ad.ß-gal and Ad.Kv4.3 (Figure 6 A–C; blots labeled as total).

Effect of Kv4.3 Overexpression on the Calcineurin Pathway
We investigated the potential role of calcineurin and its downstream transcription factor NFAT in rats subjected to pressure overload. Figure 7 shows calcineurin- and NFAT-specific Western blots performed on protein extracts of myocytes derived from sham-operated and AS hearts infected with Ad.ß-gal or Ad.Kv4.3. Calcineurin expression was significantly increased in AS myocytes derived from hearts infected with Ad.ß-gal (172.9±6.8 AU, n=4) compared with sham-operated hearts (131.8±6.7 AU, n=4 P<0.05; Figure 7A). Similarly, NFATc1 expression was also increased in AS myocytes derived from hearts infected with Ad.ß-gal (168.2±10.3 AU, n=4) compared with sham-operated hearts (120.6±4.0 AU, n=4 P<0.05; Figure 7B). Overexpression of Kv4.3 significantly reduced the expression of calcineurin by 69% (53.0±6.9 AU, n=4, P<0.05) and of NFAT by 61% (66.0±7.0 AU, n=4, P<0.05) compared with Ad.ß-gal. These results indicate that short-term delivery of the Kv4.3-expressing adenovirus is associated with inhibition of calcineurin activity in pressure-overloaded hearts.

View larger version (32K): [in this window] [in a new window]   Figure 7. Effect of in vivo adenoviral gene transfer of Kv4.3 on expression of calcineurin. Cell lysates from sham, Ad.Kv4.3–infected, and Ad.ß-gal–infected hearts were subjected to Western blotting as described for calcineurin (A) and NFATc1 (B). Quantification of calcineurin and NFAT expression was determined by densitometric scanning. There were significant decreases in protein expression of both calcineurin and NFAT in hearts transduced with Ad.Kv4.3. Data from at least 3 experiments are shown. *Ad.Kv4.3 vs Ad.ß-gal, P<0.05; sham vs Ad.ß-gal, P<0.05; sham vs Ad.Kv4.3, P<0.05. Abbreviations are as defined in text.

	Discussion

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Although changes in many ion channel currents have been reported in cardiac hypertrophy, a reduction in the density of I_to is the most prominent ionic current change resulting in APD prolongation in a process referred to as electrical remodeling.^2,7,18 It is still unclear whether APD prolongation is an adaptive or a maladaptive event of cardiac hypertrophy. Whereas I_to downregulation is frequently associated with APD prolongation regardless of the experimental animal model, it seems to occur early in the disease process,^4,18,19 which may in the short term enhance contractility of the compromised myocardium. It is conceivable that an increase in [Ca²⁺]_i modulates cardiac hypertrophy, because Ca²⁺ is an essential cofactor in the activation of several hypertrophic signaling pathways, including calcineurin and MAPKs. This is the first study demonstrating that in vivo Kv4.3 gene transfer can reverse I_to downregulation and attenuate the compensatory cardiac hypertrophy of the left ventricle under augmented loading conditions. Our findings demonstrate that short-term AS is associated with a significant reduction of Kv4.2- and Kv4.3-based I_to, APD prolongation, and cardiac hypertrophy, which is consistent with previous results.¹⁹ Overexpression of Kv4.3 was able to significantly increase the expression of Kv4.3 -subunits, increase I_to density, shorten APD, and attenuate the hypertrophic response to pressure overload.

Calcineurin
Calcineurin is a calcium/calmodulin-dependent phosphoprotein phosphatase that can dephosphorylate NFATc, resulting in its nuclear translocation and the transcriptional activation of numerous hypertrophy genes.²⁰ We report that short-term AS is associated with an increase in the expression of calcineurin and the calcineurin transcription factor NFATc1, which was restored by overexpression of Kv4.3. The role of calcineurin in the development of structural hypertrophy is well established; more recently, APD prolongation was shown to modulate this pathway. Indeed, APD prolongation secondary to reductions in I_to density was shown to influence transsarcolemmal Ca²⁺ influx through recruitment of additional L-type, voltage-dependent Ca²⁺ channels, thereby increasing cytosolic [Ca²⁺]_i in healthy and diseased myocardium. More recently, a connection between I_to reduction, APD prolongation, Ca²⁺ entry, activation of calcineurin, and cellular growth has been demonstrated in cultured neonatal ventricular myocytes infected with adenoviruses harboring dominant-negative constructs for Kv4.2.²¹ Activation of calcineurin and cellular hypertrophy were inhibited by coinfection with Ad.CAIN (calcineurin inhibitor).²¹ In this regard, enhanced Ca²⁺ transients, inotropy, and cardiac hypertrophy have been reported in transgenic mice with selective Kv4.2-based I_to reduction. Indeed, these transgenic mice exhibit significant activation of the calcineurin pathway, which may be prevented by treatment with verapamil and may explain progression of the disease from hypertrophy to failure.²² Consistent with these results, we also reported the induction of cardiac hypertrophy and disease progression to cardiac failure in transgenic mice overexpressing the 1-subunit of the L-type Ca²⁺ channel.²³ We also reported a hypercontractile phenotype, increased density of L-type calcium channels, and APD prolongation in 6- to 7-week-old calcineurin-overexpressed transgenic mice.²⁴ Interestingly, the activation of calcineurin and the onset of cardiac hypertrophy have been shown to be associated with APD prolongation secondary to an increase in L-type calcium channel density in rats subjected to AS, which was reversed with cyclosporine treatment.²⁵ Collectively, these studies suggest that activity of calcineurin may be modulated with a cellular profile of increased calcium influx, either by directly increasing the expression of L-type calcium channels or indirectly by reducing the expression of the I_to channels. Furthermore, activation of calcineurin alone exerts a feedback to increase calcium influx. This bimodal model of transsarcolemmal calcium influx and calcineurin activation may explain the commonality of these transgenic mice in disease progression and the development of heart failure at 3 to 4 months of age.

Mitogen-Activated Protein Kinases
The ERKs, JNKs, and p38 MAPKs have all been implicated in the development of cardiac hypertrophy.²⁶ We report the sustained activation of the 3 MAPK signaling pathways after AS. Activation of these pathways is known to occur as early as 5 minutes²⁷ and may be observed 8 weeks after AS. A relation between calcium influx via L-type calcium channels, activation of MAPKs,²⁸ and alterations in gene expression has already been demonstrated in neuronal cells and mammalian cell lines.²⁹ We found that overexpression of Kv4.3-based I_tos did not alter the activation of these kinases, suggesting that there is no association between I_to density, membrane potential, and MAPK activation. However, it is not clear whether the lack of effect may be due to the fact that <30% of the myocardium was infected in vivo and that a higher infectivity may be required to modulate the activity of these kinases. It is also quite possible that the apparent effect of I_to on MAPKs is inhibited or antagonized by more dominant mechanisms in the physiological setting, where many agonists act in concert to remodel the myocardium. Nevertheless, it remains to be determined whether I_to suppression would have any effect on the activation of MAPKs.

Contractility
At 4 mmol/L Ca²⁺, there was significantly enhanced contractility in terms of peak intracellular Ca²⁺ and percent shortening, whereas no significant changes were observed in the in vivo setting. In early hypertrophy (1 to 2 weeks after aortic constriction), as the ventricle is adapting to the increased load, cardiac myocytes hypertrophy, and with prolonged AP, more calcium moves through the sarcolemma and an increased sarcoplasmic reticulum calcium release is observed. A number of studies have also related this phenomenon of enhanced peak calcium in isolated cells from early hypertrophied hearts.³⁰ Other studies in early hypertrophy have also shown enhanced contractility in rat hearts after short-term pressure overload.³¹ In general, in vivo measurements have not shown enhanced contractility, even though some studies did show increased fractional shortening.³² Our own previously published studies have shown also that short-term pressure overload did not enhance in vivo contractile function.³³ Even though each myocyte forms the functional basis for ventricular function, in our experiments, single cardiomyocytes are unloaded and in this setting, the contractile parameters cannot be extrapolated to the loaded state of the ventricle in the body.

Limitations of This Study
This study has demonstrated that overexpression of Kv4.3-based I_tos abrogates the hypertrophic response in the short term. It remains to be determined whether long-term infection with Ad.Kv4.3 would significantly modify I_to expression and calcineurin levels. We and others^4,10,18,23 reported that modulation of early repolarization is an important determinant of L-type calcium channel activity and calcium influx in the myocardium. It is unknown whether these results may be transferable to the diseased human myocardium. Rodents possess a characteristic triangular short AP because of prominent expression of I_tos, whereas larger mammalian species and humans possess a much smaller I_to density and an AP profile with a spike-dome morphology. These data are interesting and may not be extrapolated to heart failure patients because of the presence of comorbid conditions and large differences in AP profile. It is interesting to note that patients with long Q-T syndrome, which has been attributed to mutations of the delayed rectifier (rapid and slow component) K⁺ channel, do not develop ventricular hypertrophy. One possible explanation may be the fact that these channels operate toward the end of cardiac repolarization, during which time calcium channels have already been rendered inactive.

The results in the present study support the hypothesis that cardiac hypertrophy, downregulation of I_to, and APD prolongation occur early after pressure overload and that in vivo cardiac gene transfer of Kv4.3-based I_to can increase I_to density, shorten APD, and attenuate the hypertrophic responses via a calcineurin pathway.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health: HL 57623, HL 71763 (to R.J.H.), HL 49574 (to J.K.G.), 5 T32 HL07382 (to A.S.), P01 22619 (to A.S.), HL076659 (to D.L.), and HL 69842 and a Beeson Scholar Award to R.J.H. from the American Federation of Aging Research. R.K. was supported by a research fellowship award from the Heart and Stroke Foundation of Canada.

	Footnotes

 
*The first 2 authors contributed equally to this work.

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