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Originally published In Press as doi:10.1074/jbc.M607774200 on October 23, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38498-38506, December 15, 2006
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Calcineurin Increases Cardiac Transient Outward K+ Currents via Transcriptional Up-regulation of Kv4.2 Channel Subunits*

Nanling Gong{ddagger}§1, Ilona Bodi, Carsten Zobel||, Arnold Schwartz, Jeffery D. Molkentin**2, and Peter H. Backx, Career Investigator of the Heart and Stroke Foundation of Ontario{ddagger}§3

From the {ddagger}Departments of Physiology and Medicine, Heart and Stroke/Richard Lewar Centre of Excellence, and the §Division of Cardiology, University Health Network, University of Toronto, Toronto, Ontario M5S 3E2, Canada, the Institute of Molecular Pharmacology and Biophysics, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, the ||Department of Internal Medicine III, University of Cologne, Cologne 50924, Germany, and the **Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio 45229

Received for publication, August 14, 2006 , and in revised form, September 27, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fast transient outward potassium currents (Ito,f) are critical determinants of regional heterogeneity of cardiomyocyte repolarization as well as cardiomyocyte contractility. Additionally, Ito,f densities are markedly down-regulated in cardiac hypertrophy and heart disease, conditions associated with activation of the serine/threonine phosphatase calcineurin (Cn). In this study, we investigated the regulation of Ito,f expression by Cn in cultured neonatal rat ventricular myocytes (NRVMs) with and without {alpha}1-adrenoreceptor stimulation with phenylephrine (PE). Overexpression of constitutively active Cn in NRVMs induced hypertrophy and caused profound increases in Ito,f density as well as Kv4.2 mRNA and protein expression and promoter activity, without affecting Kv4.3 or KChIP2 levels. The effects of Cn on hypertrophy, Ito,f, and Kv4.2 transcription were associated with NFAT activation and were abrogated by NFAT inhibition. Despite activating Cn and inducing hypertrophy in NRVMs, PE resulted in profound down-regulation of Ito,f densities as well as Kv4.2, Kv4.3, and KChIP2 expression. Although hypertrophy and NFAT activation were inhibited by the Cn inhibitory peptide CAIN, Ito,f and Kv4.2 expression were further reduced by CAIN, whereas Cn overexpression eliminated PE-induced reductions in Ito,f and Kv4.2 expression without affecting Kv4.3 or KChIP2 levels. We conclude that Cn increases cardiac Ito,f densities by positively regulating Kv4.2 gene transcription. Consistent with this conclusion, we found that Ito,f was increased in myocytes isolated from young mice overexpressing Cn prior to the development of heart disease. This positive regulation of Kv4.2 transcription by Cn activation is expected to minimize the reductions in Ito,f and Kv4.2 expression observed in hypertrophic cardiomyocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal cardiac repolarization is orchestrated by coordinated activity of many K+ currents, which vary between regions of the heart as well as between different species. One K+ current of particular interest in most mammalian species, including humans, is the cardiac transient outward K+ current, which is composed of fast (Ito,f)4 and slow types. Ito,f is encoded by pore-forming {alpha}-subunits (i.e. Kv4.2 and Kv4.3 in rodents and Kv4.3 in humans) and auxiliary KChIP2 subunits. Variations in Ito,f as a result of regional differences in Kv4.2 (rodents) (1, 2) or Kv4.3 or KChIP2 (humans and dogs) (3, 4) are major contributors to electrical heterogeneity of repolarization in the heart, which is strongly linked to arrhythmias, particularly the Brugada syndrome (5). Ito,f densities also have profound effects on the strength and timing of myocyte contractions (6, 7).

Reductions in Ito,f as well as Kv4.2, Kv4.3, and KChIP2 expression are commonly observed in conjunction with action potential duration prolongation following treatment of cardiomyocytes with hypertrophic agents such as {alpha}1-adrenoreceptor ({alpha}1AR) agonists (8) and angiotensin II receptor agonists (9) as well as in cardiac hypertrophy (10-12) and heart disease (13-15). The mechanisms responsible for altered Ito,f levels induced by {alpha}1AR stimulation in myocytes (8) as well as in heart disease (13-15) are unclear. Recent studies concluded that calcineurin (Cn), a Ca2+/calmodulin-activated serine/threonine phosphatase that dephosphorylates NFATc1-4 (16), causes reductions in Ito,f (17, 18). This is a particularly attractive mechanism because Cn is necessary and sufficient to induce hypertrophy in rodents with pressure overload (19, 20) as well as in myocytes treated with {alpha}1AR agonists (21, 22). However, K+ currents are reported to be unchanged in young mice with cardiac overexpression of Cn, despite significant hypertrophy prior to the development of cardiomyopathy (23). Moreover, Ito,f is increased in NFATc4 transgenic mice (24). Thus, despite the importance of Ito,f and Cn in heart disease, the precise role of Cn in the regulation of Ito,f remains poorly understood.

Our experiments were designed to elucidate the regulation of Ito,f by Cn in neonatal rat ventricular myocytes (NRVMs). Our results establish that Cn overexpression strongly enhanced Ito,f and Kv4.2 transcription in an NFAT-dependent manner. In addition, Cn overexpression reversed Ito,f reductions induced by {alpha}1AR stimulation with phenylephrine (PE) via increases in Kv4.2 mRNA and protein, whereas Cn inhibition with the Cn inhibitory peptide CAIN (21) caused further reductions in Ito,f and Kv4.2 transcription observed with PE treatment. These results demonstrate that Cn/NFAT is a positive regulator of Ito,f via transcriptional up-regulation of Kv4.2 in myocytes and thus minimizes the degree of Ito,f reductions induced in cardiac hypertrophy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation, Culture, and Intervention of NRVMs—Day 1 or 2 Sprague-Dawley NRVMs (Charles River Laboratories, Inc., Montreal, Canada) were isolated and cultured in medium containing 5% fetal bovine serum in the presence of 0.1 mmol/liter bromodeoxyuridine (Sigma) following removal of non-myocytes by pre-plating (37 °C, 1 h) as described previously (25). After 24 h, the myocytes were cultured in the absence of serum (and supplemented with insulin-transferrin-selenium-X (Invitrogen), 25 µg/ml ascorbic acid, and 1 nmol/liter LiCl) and infected with adenoviruses (green fluorescent protein (GFP), CAIN, constitutively active Cn, {Delta}NFATc3, or VIVIT (a peptide consisting of Met-Ala-Gly-Pro-His-Pro-Val-Ile-Val-Ile-Thr-Gly-Pro-His-Glu-Glu designed to inhibit Cn-mediated NFAT activation (32); VIVIT adenoviruses were kindly provided by Dr. S. D. Kraner) at a multiplicity of infection of 5-10 for 16 h. Myocyte cultures were then washed and incubated in the presence or absence of the {alpha}1AR agonist PE (100 µmol/liter) for 48 h. Infection efficiency was typically >98%, with minimal cell death at these levels of multiplicity of infection. Myocytes were plated on laminin-coated coverslips at a density of 1.5 x 105 cells/ml for experiments involving immunofluorescence staining and electrophysiological recordings and at 5 x 105 cells/ml for all other experiments.

Immunofluorescence Staining of NRVMs—Immunoconfocal fluorescence was used to assess myocyte size and numbers. To perform these experiments, cultured cells were fixed and permeabilized with methanol (-20 °C, 15 min). After washing with phosphate-buffered saline, cells were incubated with 1:200 anti-{alpha}-actinin monoclonal antibody (sarcomeric; Sigma) in phosphate-buffered saline with 1% bovine serum albumin at room temperature for 1 h. Phosphate-buffered saline-washed cells were subsequently incubated at room temperature for 30 min with 1:400 Alexa Fluor® 488-labeled goat anti-mouse IgG antibody (H + L; Molecular Probes) and viewed under a confocal microscope. Typically, >98% of the cells were {alpha}-actinin-positive 96 h after NRVM isolation, confirming the dominance of myocytes in the culture.

[3H]Leucine Uptake Experiments—The rate of protein synthesis was measured by [3H]leucine uptake experiments. Cells were cultured in leucine-free Dulbecco's modified Eagle's medium/nutrient mixture F-12 (Sigma) in the presence of 1 µCi/ml [3H]leucine with or without 100 µmol/liter PE for 48 h. Cells were then incubated with 10% trichloroacetic acid at 4 °C for 2 h, followed by cell lysis with 1 N NaOH. Each of the lysed samples was added to 7 ml of ScintiSafeTM 30% liquid scintillation mixture (Fisher) in a 20-ml polyethylene scintillation vial (Fisher) and subjected to liquid scintillation counting.

Electrophysiological Recordings in NRVMs—Whole-cell voltage-clamp recordings were done as described previously (8, 25) at room temperature. Whole-cell currents were filtered at 2 kHz (Axon 200A amplifier). Myocytes were perfused with drug-free extracellular solution for at least 15 min before measurements were performed. The extracellular solution contained 140 mmol/liter NaCl, 4 mmol/liter KCl, 2 mmol/liter CaCl2, 1 mmol/liter MgCl2, 0.5 mmol/liter CdCl2, 10 mmol/liter HEPES, and 10 mmol/liter glucose (pH 7.4). The intracellular solution contained 140 mmol/liter KCl, 1 mmol/liter MgCl2, 10 mmol/liter EGTA, 10 mmol/liter HEPES, and 5 mmol/liter MgATP (pH 7.25).

Chord conductance (G) was calculated according to the following equation: G = I/(Vm - EK), where I is Ito,f recorded in response to step depolarization to Vm and EK represents the Nernst potential for K+ ions. Fits of the relationship between G and Vm to the Boltzmann equation allow estimation of the maximal chord conductance (Gmax) and the voltage for half-maximal activation of Ito,f (i.e. V1/2). Slope conductance (Gslope) was estimated by linear regression of the relationship between peak Ito,f and Vm at step voltages ranging from +10 to +60 mV.

Electrophysiological Recordings in Cardiomyocytes from Young Mouse Hearts—Cardiomyocytes were dissociated from the ventricular apices of 3-4-week-old non-transgenic and Cn-overexpressing transgenic mice according to the technique described previously (23). Whole-cell voltage-clamp recordings were done as described previously (26) at room temperature. Cardiomyocytes were perfused with normal Tyrode's solution containing 138 mmol/liter NaCl, 4 mmol/liter KCl, 2 mmol/liter CaCl2, 1 mmol/liter MgCl2, 10 mmol/liter glucose, 10 mmol/liter HEPES, and 0.33 mmol/liter NaH2PO4 (adjusted to pH 7.4 with NaOH). L-type Ca2+ current was eliminated by 0.3 mmol/liter CdCl2 included in the recording solution. The pipette solution contained 120 mmol/liter potassium glutamate, 10 mmol/liter KCl, 2 mmol/liter MgCl2, 10 mmol/liter HEPES, 5 mmol/liter EGTA, and 2 mmol/liter MgATP (adjusted to pH 7.2 with KOH).

RNA Extraction and Quantitative Real-time PCR Analysis— Total RNA was extracted from NRVMs using TRIzol reagent (Invitrogen) as recommended by the manufacturer and treated with RNase-free DNase I. cDNA was synthesized from 2 µg of total RNA using SuperScriptTM (Invitrogen) and amplified by real-time PCR (Applied Biosystems). For KChIP2, TaqMan probe and primers (assay ID Rn01411445_g1) were used for mRNA quantification. Kv4.2, Kv4.3, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified using SYBR Green PCR Master Mix (Applied Biosystems). The primers for Kv4.2 were 5'-GTGTCAGGAAGTCATAGAGGC-3' (forward) and 5'-TTACAAAGCAGACACCCTGA-3' (reverse). The primers for Kv4.3 were 5'-CACCACCTGCTACACTGCTTAGAA-3' (forward) and 5'-TCTGCTCATCAATAAACTCGTGGTT-3' (reverse). The primers for GAPDH were 5'-TGCACCACCAACTGCTTAG-3' (forward) and 5'-GATGCAGGGATGATGTTC-3' (reverse). mRNA determinations were done in duplicate using standard curves, and specificity was confirmed using 2% agarose gel and/or melting curves.

Western Immunoblot Analysis—Cells were collected and lysed in Laemmli buffer. Equal amounts of protein (determined using the Bio-Rad RC DC assay) were loaded, separated on 12% SDS-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes, and probed using rabbit polyclonal antibodies against Kv4.2 or Kv4.3 (Chemicon) or KChIP2 (Affinity BioReagents). GAPDH was detected by a mouse monoclonal antibody (Research Diagnostics, Inc.). Anti-rabbit or anti-mouse antibodies conjugated with horseradish peroxidase (Amersham Biosciences) were used as secondary antibodies to allow protein visualization with enhanced chemiluminescence (Amersham Biosciences).

Isolation of the KChIP2 Promoter and Cloning into the pGL3-Basic Vector—Rat genomic DNA was isolated from Sprague-Dawley rat livers using previously described methods (27). The KChIP2 promoter region (-1524 to +312) was isolated from rat genomic DNA by AdvantageTM-GC genomic PCR (BD Biosciences) using primers designed according to the nucleotide sequence of NW_043419. The primers used were 5'-TAAGCTAGCTGTGGGGTGCATCATCTCTATTCA-3' (forward) with the addition of an NheI site and 5'-AACCCCGGGAACTATCACCGACTCACCCGTAAG-3' (reverse) with the addition of an SmaI site at the 5'-end. Following purification, the amplified DNA was digested with NheI and SmaI and subcloned into the pGL3-Basic vector (Promega Corp.). Positive clones containing the KChIP2 promoter were confirmed by nucleotide sequencing and Dual-Luciferase assay.

Transfection and Promoter Activity Measurements—For promoter experiments, myocytes were transfected 16 h after viral infection with pGL3 with a firefly luciferase reporter gene driven by the Kv4.2 (kindly provided by Dr. K. Takimoto) (28), Kv4.3 (kindly provided by Dr. E. S. Levitan) (29), or KChIP2 promoter using Lipofectamine 2000 (Invitrogen). pRL-TK encoding Renilla luciferase was cotransfected as a control to correct for transfection efficiency. Cell lysates were obtained 48 h after transfection to measure Dual-Luciferase activity (Promega Corp.). In the experiments to measure the activation of Cn/NFAT, NRVMs were transfected with pGL3 with a firefly luciferase reporter gene driven by a promoter containing three NFAT cis-elements (kindly provided by Dr. A. Rao).

Statistical Analysis—All data are expressed as the means ± S.E. Statistical significance was calculated using Student's t test to compare two groups and analysis of variance to compare multiple groups. p values <0.05 were considered statistically significant.


Figure 1
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  		FIGURE 1.
Typical immunofluorescence images, cell capacitance and [3H]leucine uptake data (A), K+ currents (B and C), and chord conductance (D) obtained from NRVMs overexpressing GFP or Cn. A, images of myocytes stained with {alpha}-actinin as described under "Experimental Procedures." Cell capacitance was estimated by integrating the area under an uncompensated capacity transient elicited by a 10-mV test pulse (25 ms) from a holding potential of -80 mV. Data are presented as the means ± S.E. (n > 16 for capacitance measurement and n = 4 for [3H]leucine uptake experiments). B, representative families of outward potassium current density recorded in myocytes. Currents were recorded in response to step depolarization from -40 to +60 mV (increments of 10 mV) for 450 ms from a holding potential of -80 mV in myocytes. A brief pre-pulse (50 ms) to -40 mV was applied to eliminate activated sodium current. Ito,f density (pA/pF) was determined by dividing Ito,f by the cell capacitance, where Ito,f was estimated as the difference between the peak and the sustained outward currents. C, Ito,f density plotted as a function of the step potential. D, chord conductance (G) plotted as a function of the step potential after fitting data to the Boltzmann equation. Error bars represent the means ± S.E. of six cells/group. *, p < 0.05 versus GFP.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cn Increases Ito,f via NFAT-dependent Increases in Kv4.2 Transcription—To examine the regulation of Ito,f by Cn, we overexpressed constitutively active Cn in NRVMs. As reported previously (30, 31), Cn overexpression induced marked myocyte hypertrophy as determined by cell microscopy, cell capacitance data, and [3H]leucine uptake experiments (Fig. 1A). Rather than decreasing Ito,f, as might be expected from previous studies (17, 18), Cn caused a 2-fold increase in Ito,f density, which was quantified as the difference in the peak current minus the sustained current (Isus) at the end of the pulse divided by the cell capacitance. Ito,f density recorded at +60 mV (Fig. 1, B and C; and Table 1) was increased in myocytes overexpressing Cn compared with control myocytes. As expected, Gslope, which gives a more direct measure of the extent of Ito,f enhancement, was also increased in myocytes overexpressing Cn compared with control myocytes, along with increased Gmax of Ito,f (Table 1). The increases in Ito,f density and conductance were not accompanied by shifts (p = 0.57) in the voltage dependence of Ito,f activation as assessed by estimates of the voltages required for Ito,f to reach 50% of the maximal conductance (V1/2) (data not shown). In addition, there were no changes in the time course of Ito,f inactivation (data not shown). The effect of Cn overexpression on Ito,f appeared to be specific because the current density remaining at the end of the voltage step (i.e. Isus) was not changed (p > 0.50) in myocytes by overexpression of Cn (Table 1).


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  		TABLE 1
Electrophysiological properties of Ito,f and Isus recorded in NRVMs Data are the means ± S.E. The values were calculated from the current recordings illustrated in Figs. 1 and 4. Gslope is estimated from the slope conductance of Ito,f (between +10 and +60 mV). Gmax is the maximal conductance estimated from plots of Ito,f/(Vm – EK) (see "Experimental Procedures"). n indicates the number of cells studied.

 
To test whether the increases in Ito,f induced by Cn were associated with changes in the expression of genes encoding Ito,f, we measured the expression of Kv4.2, Kv4.3, and KChIP2. As shown in Fig. 2, Cn overexpression increased Kv4.2 mRNA and protein, without affecting Kv4.3 or KChIP2 expression compared with control cells. To investigate whether the increases in Kv4.2 mRNA were related to increased transcription activity (versus changes in mRNA stability), NRVMs were transfected with Kv4.2 (28), Kv4.3 (29), and KChIP2 promoter constructs. Fig. 3A shows that overexpression of Cn increased the activity of the Kv4.2EB promoter (-3162 to +592). This increase in Kv4.2EB promoter activity, as well as the changes in Ito,f, appeared to be mediated by NFAT dephosphorylation because Ito,f recorded at +60 mV in NRVMs upon overexpression of Cn was reduced to 8.71 ± 2.25 pA/picofarad (pF) (n = 6) when co-infected with adenoviral VIVIT (32), a selective inhibitory peptide of Cn-mediated NFAT dephosphorylation. VIVIT overexpression alone did not alter Ito,f (11.52 ± 1.86 pA/pF, n = 5). Cn-mediated Kv4.2 promoter activity was abolished by coinfection with VIVIT, which alone had no effect on basal Kv4.2 promoter activity (Fig. 3A), and Cn overexpression induced a 6-fold increase in NFAT activity (Fig. 3B), whereas overexpression of {Delta}NFATc3 (i.e. constitutively active NFATc3) (33) stimulated Kv4.2EB promoter activity by similar amounts (Fig. 3A). By contrast, neither Cn nor {Delta}NFATc3 overexpression altered the activity of the Kv4.3 (-2337 to +54), Kv4.3 (-663 to +54), or KChIP2 (-1524 to +312) promoter (data not shown), consistent with mRNA and protein results (Fig. 2). These results establish that Cn increases Ito,f via NFAT-dependent increases in Kv4.2 transcription.

Cn Minimizes Ito,f Reductions following Chronic {alpha}1AR Stimulation—Because several previous studies have suggested that Cn activation in cardiac hypertrophy is responsible for reductions in Ito,f (8, 21), we investigated the regulation of Ito,f by Cn in an in vitro hypertrophic model induced by {alpha}1AR stimulation. To achieve this, we overexpressed either CAIN or constitutively active Cn in NRVMs in the presence of 100 µmol/liter PE for 48 h. As expected from a previous study (8), PE treatment induced marked myocyte hypertrophy based on cell microscopy, cell capacitance data, and [3H]leucine uptake experiments (Fig. 4A) and increased NFAT activity by 4-fold compared with myocytes not treated with PE (Fig. 4B). In addition, overexpression of CAIN (21), a specific inhibitory peptide of Cn, inhibited NFAT activation and prevented hypertrophy following treatment with PE, confirming the essential role of Cn signaling in {alpha}1AR-induced hypertrophy. Consistent with previous studies (8, 34), Fig. 4 (C and D) and Table 1 show that Ito,f densities along with Gslope and Gmax were decreased in PE-treated myocytes compared with control myocytes without PE. However, contrary to expectations based on studies concluding that Cn is responsible for Ito,f reductions (17, 18), CAIN overexpression in PE-treated myocytes further reduced Ito,f densities, Gslope, and Gmax (Fig. 4, C and D; and Table 1). On the other hand, CAIN overexpression did not alter (p > 0.40) the basal levels of Ito,f densities, Gslope, or Gmax (data not shown). Remarkably, as shown in Fig. 4 (C and D) and Table 1, overexpression of constitutively active Cn (31) completely reversed the PE-induced reductions in Ito,f densities as well as Gslope and Gmax, without affecting the activation-gating properties of Ito,f as assessed by estimates of V1/2. Neither PE treatment nor Cn overexpression altered the kinetics of Ito,f inactivation. The effects of Cn and CAIN overexpression on Ito,f appeared to be specific because Isus density was not changed (p > 0.50) in PE-treated myocytes by overexpression of Cn or CAIN (Table 1).


Figure 2
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  		FIGURE 2.
Quantitation of mRNA levels (A) and representative images and quantitation of protein levels (B) of Kv4.2, Kv4.3, and KChIP2 in NRVMs overexpressing GFP or Cn. RNA extracted from these myocytes was subjected to reverse transcription and subsequent real-time PCR to measure mRNA levels by the standard curve method. Equal amounts of collected protein samples were subjected to Western immunoblot analysis. GAPDH was used as an internal control. The mRNA or protein level normalized to the corresponding GAPDH level in NRVMs infected with adenoviral GFP was set as 100. Data are presented as the means ± S.E. (n = 5 for mRNA and n = 3 for protein). *, p < 0.05 versus GFP.

 


Figure 3
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  		FIGURE 3.
Activity of the Kv4.2EB promoter in NRVMs overexpressing GFP, Cn, {Delta}NFATc3, or VIVIT (A) and activity of a promoter containing three NFAT cis-elements in NRVMs overexpressing GFP or Cn (B). Sixteen hours after viral infection, cells were transfected with pGL3 with the firefly luciferase gene driven by the Kv4.2EB promoter or a promoter containing three NFAT cis-elements. pRL-TK encoding Renilla luciferase was cotransfected as a control for transfection efficiency. Cell lysates were obtained 48 h after transfection and subjected to Dual-Luciferase assay. Firefly luciferase activity normalized to Renilla luciferase activity is plotted for each category of NRVMs. The basal activity of the promoter in NRVMs infected with adenoviral GFP was artificially set as 1. Error bars represent the means ± S.E. (n ≥ 3). *, p < 0.05 versus GFP.

 
In agreement with the changes in Ito,f density, PE reduced Kv4.2, Kv4.3, and KChIP2 mRNA levels as summarized in Fig. 5, with similar reductions in Kv4.2, Kv4.3, and KChIP2 protein levels compared with the control (Fig. 6). Overexpression of CAIN had no effect on the basal mRNA and protein levels of Kv4.2, Kv4.3, and KChIP2 (data not shown), but further reduced Kv4.2 mRNA and protein expression induced by PE treatment, without affecting Kv4.3 and KChIP2 expression (Figs. 5 and 6). On the other hand, overexpression of Cn following PE treatment increased Kv4.2 mRNA and protein levels compared with the control while having no effect (p > 0.12) on Kv4.3 and KChIP2 mRNA and protein levels (Figs. 5 and 6). It is important to note that the differences in Kv4.3 mRNA between PE and CAIN/PE or between PE and Cn/PE as shown in Fig. 5B are not significant. At first glance, it may seem surprising that the level of Ito,f in the PE-treated myocytes expressing Cn was not increased (p = 0.53) above that in the control (i.e. myocytes not treated with PE) given that Cn induced a nearly 2-fold increase in Kv4.2 protein expression above the control when overexpressed in PE-treated myocytes. However, this lack of concordance between Ito,f levels and Kv4.2 protein levels is consistent with the large reductions in Kv4.3 and KChIP2 expression because rodent Ito,f channels are formed primarily as heterotetramers of Kv4.2 and Kv4.3 channels (1) and because Ito,f channels require KChIP2 for membrane insertion of functional channels (35). Therefore, taken together, these results establish that Cn is a positive regulator of Kv4.2 expression and Ito,f density when Ito,f is reduced by PE.


Figure 4
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  		FIGURE 4.
Shown are typical immunofluorescence images and cell capacitance and [3H]leucine uptake data (A) and K+ currents (B) obtained from NRVMs overexpressing GFP, CAIN, or Cn following PE treatment. The activity of a promoter containing three NFAT cis-elements was enhanced in NRVMs treated with PE. In C, representative families of outward potassium current density are recorded. The voltage protocols were as described in the legend to Fig. 1. In D, Ito,f density-voltage relationships are displayed as described in the legend to Fig. 1. Cell capacitance (n > 16), [3H]leucine uptake data (n = 4), and Gslope (between +10 and +60 mV; n = six cells/group) were compared by analysis of variance analysis between groups. *, p < 0.05 versus GFP; {gamma}, p < 0.05 versus GFP + PE. CTL, control.

 
The above results establish that Ito,f is positively regulated by Cn in cultured neonatal myocytes. To explore whether Cn also regulates Ito,f in intact myocardium, we measured Ito,f in myocytes isolated from mice overexpressing Cn. We performed these experiments in mice that were 3-4 weeks of age to reduce the complexities associated with cardiac hypertrophy and heart disease that become apparent in older mice (30). As summarized in Fig. 7 and Table 2, myocytes isolated from mice overexpressing Cn in the heart had higher Ito,f densities and Gslope than age-matched non-transgenic littermate control mice. These results demonstrate that Ito,f is positively regulated by Cn in intact adult myocardium.


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  		TABLE 2
Electrophysiological properties of Ito,f recorded in cardiomyocytes dissociated from the ventricular apices of 3–4-week-old non-transgenic and Cn-overexpressing transgenic mice Data are the means ± S.E. The values were calculated from the current recordings illustrated in Fig. 7. Gslope is estimated from the slope conductance of Ito,f (between +10 and +60 mV). n indicates the number of cells studied.

 


Figure 5
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  		FIGURE 5.
Quantitation of mRNA levels of Kv4.2 (A), Kv4.3 (B), and KChIP2 (C) in NRVMs overexpressing GFP, CAIN, or Cn in the presence of PE. Sixteen hours after viral infection, NRVMs were treated with 100 µmol/liter PE for 48 h. RNA extracted from these myocytes was subjected to reverse transcription and subsequent real-time PCR to measure mRNA levels by the standard curve method. GAPDH was used as an internal control. The mRNA level normalized to the GAPDH level in NRVMs infected with adenoviral GFP (control (CTL)) was set as 100. Data are presented as the means ± S.E. (n = 5). *, p < 0.05 versus CTL; {gamma}, p < 0.05 versus PE.

 


Figure 6
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  		FIGURE 6.
Representative images and quantitation of protein levels of Kv4.2 (A), Kv4.3 (B), and KChIP2 (C) in NRVMs overexpressing GFP, CAIN, or Cn in the presence of PE. Sixteen hours after viral infection, NRVMs were treated with 100 µmol/liter PE for 48 h. Equal amounts of collected protein samples were subjected to Western immunoblot analysis. GAPDH was used as an internal control. The protein level normalized to the GAPDH level in NRVMs infected with adenoviral GFP (control (CTL)) was set as 100. Data are presented as the means ± S.E. *, p < 0.05 versus CTL; {gamma}, p < 0.05 versus PE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our study has confirmed that overexpression of constitutively active Cn induces hypertrophy. Surprisingly, the Cn-induced hypertrophy in cultured NRVMs was associated with marked increases in Ito,f that correlated strongly with increases in the mRNA, protein, and promoter activities of Kv4.2, but not the other molecular constituents of Ito,f (i.e. Kv4.3 and KChIP2). The increases in Kv4.2 mRNA were associated with enhanced promoter activity, suggesting that Cn induces increases in Kv4.2 transcription activity versus altered mRNA stability. Positive regulation of Ito,f by Cn was also observed in very young (3-4 weeks old) mice overexpressing Cn, suggesting that this positive regulation of Ito,f is not specific to cultured neonatal myocytes.


Figure 7
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  		FIGURE 7.
Typical K+ currents obtained from cardiomyocytes dissociated from the ventricular apices of 3-4-week-old non-transgenic (NTG) and Cn-overexpressing transgenic (Cn TG) mice. A, representative families of outward potassium currents recorded in dissociated cardiomyocytes in response to step depolarization from -40 to +60 mV (increments of 10 mV) for 4000 ms from a holding potential of -80 mV. Ito,f was estimated by fitting the decay phase of outward K+ currents with bi- or triexponential function using Clampfit Version 9.0, and Ito,f density (pA/pF) was determined by dividing Ito,f by the cell capacitance (Cm). B, Ito,f density plotted as a function of the step potential. Gslope values (between +10 and +60 mV) were compared by Student's t test between groups. *, p < 0.05 versus NTG.

 
Consistent with previous studies concluding that the hypertrophy induced by Cn requires dephosphorylation of NFAT (probably NFATc3 and NFATc4) (30, 36), which leads to nuclear translocation and transcriptional alterations in gene expression (30), NFAT inhibition (with VIVIT) blocked both the Cn-induced hypertrophy and the enhancement of Ito,f and Kv4.2 promoter activity. On the other hand, changes in NFAT did not affect Kv4.3 or KChIP2 promoter activity.5 These results establish that Cn activation increases Ito,f via transcriptional enhancement of Kv4.2, but not Kv4.3 or KChIP2. Our findings also suggest that Cn/NFAT has no effect on Kv4.2 transcription or Ito,f under basal conditions because VIVIT alone had no effect on Ito,f or Kv4.2 promoter activity, whereas CAIN alone did not alter Ito,f or Kv4.2 expression.

It is important to note that only the 3.5-kb Kv4.2EB promoter (-3162 to +592) activity was increased by Cn and {Delta}NFATc3, whereas the shorter promoters Kv4.2HB (-1094 to +592) and Kv4.2SB (-432 to +592) (28) were not activated by Cn or {Delta}NFATc3. These observations are consistent with the existence of 13 consensus NFAT-binding sites (i.e. GGAAA cis-elements) between -3162 and -1095 in the Kv4.2 promoter versus only three NFAT-binding sites between -1094 and 0. Similarly, the inability of Cn or {Delta}NFATc3 to increase Kv4.3 or KChIP2 transcription correlates with the presence of only six putative NFAT sites in the Kv4.3 promoter (-2337 to +54) and five NFAT sites in the KChIP2 promoter (-1524 to +312). Although the ability of Cn and NFAT to increase Kv4.2 promoter activity correlates with the number of NFAT-binding sites, NFAT activation can be modulated by other transcription factors (37), which could also conceivably contribute to or be responsible for the enhanced Kv4.2 transcription by Cn.

We further investigated the role of Cn in the regulation of Ito,f in an in vitro hypertrophic model of cardiomyocytes induced by {alpha}1AR stimulation. Our experiments confirmed that chronic {alpha}1AR stimulation in cultured NRVMs decreased Ito,f in association with reductions in Kv4.2, Kv4.3, and KChIP2 mRNA and protein levels (8, 34). We have also demonstrated that Cn inhibition (with CAIN overexpression) blocked {alpha}1AR-mediated hypertrophy in cultured NRVMs, as reported previously (21). Previous studies established that hypertrophy induced by {alpha}1AR stimulation in cultured NRVMs is associated with Cn activation (8, 21) and Ito,f reductions (8). Similarly, Cn activation (19, 38, 39) and Ito,f reductions (10, 11, 14, 18) are commonly observed in various animal models of cardiac hypertrophy and heart disease as well as in heart disease patients (40, 41). Collectively, these observations have led to the suggestion that Cn activation is responsible for the reductions in Ito,f (17, 18) as well as hypertrophy. However, rather unexpectedly (17, 18), our results showed that, in cultured NRVMs chronically treated with {alpha}1AR agonists, inhibition of Cn (using CAIN) caused further reductions in Ito,f, despite blocking hypertrophy, whereas overexpression of constitutively active Cn induced large increases in Ito,f density without inhibiting myocyte hypertrophy. These changes in Ito,f were associated with changes in mRNA and protein expression of Kv4.2, but not Kv4.3 or KChIP2, a pattern identical to that observed in the absence of {alpha}1AR stimulation. Thus, in the presence of cardiac hypertrophy, we conclude that Cn activation will minimize the degree of reductions in Ito,f by driving Kv4.2 transcription (see the proposed model in Fig. 8).

In contrast to our findings, a previous study showed that inhibition of Cn with cyclosporin or knock out of NFATc3 prevents Ito,f reductions induced by myocardial infarction in adult mice (18). The differences between our experiments using NRVMs and those using infarcted (18) or normal (42) adult mouse hearts may reflect age-dependent differences in signaling or differences between cultured isolated myocytes and intact myocardium. Alternatively, because the reductions in Ito,f following myocardial infarction in adult mice also require betaAR stimulation (18), it is plausible that betaAR and {alpha}1AR stimulation activates distinct signaling pathways capable of modulating the net effects of Cn. The potential differential Cn-mediated regulation of Ito,f activated by {alpha}1AR and betaAR stimulation is in keeping with the complex regulation of Cn signaling in the myocardium. For example, Cn/NFAT-mediated tumor necrosis factor-{alpha} expression and cardiomyocyte hypertrophy are suppressed by interaction between HDAC4 and the HSP40 protein Mrj (33).


Figure 8
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  		FIGURE 8.
Proposed model for the regulation of Ito,f by Cn in NRVMs. {alpha}1AR stimulation reduces expression of Kv4.2, Kv4.3, and KChIP2 while also activating Cn, leading to myocyte hypertrophy. Cn activation also causes an increase in Ito,f via increased levels of Kv4.2 mRNA and protein, which will minimize Ito,f reductions induced by {alpha}1AR stimulation. The dashed arrow shows pathways not yet determined, whereas the solid arrows show the pathways determined in this study.

 
The reductions in Ito,f in failing hearts from Cn transgenic mice (23, 43) also suggest that Cn activation reduces Ito,f. However, because heart disease causes Ito,f reductions (13-15) and because these Cn transgenic mice develop heart disease by 6 weeks of age (30), the interpretation of these studies requires caution. Consistent with Ito,f being reduced as a consequence of heart disease induced by Cn in transgenic mice, Kv4.2 expression and Ito,f were reported to be unchanged in younger mice (6-7 weeks of age) despite marked hypertrophy (23). Consequently, we investigated Ito,f in 3-4-week-old Cn transgenic mice. Consistent with our cultured myocyte results, Ito,f is indeed elevated in young mice overexpressing Cn, establishing that Cn is also a positive regulator of Ito,f in intact adult myocardium.

A key conclusion of our study is that Cn activation is not responsible for the reductions in Ito,f or Kv4.2, Kv4.3, and KChIP2 expression induced by {alpha}1AR stimulation in NRVMs. Moreover, Cn overexpression in PE-treated myocytes increased Ito,f levels, but only back to the levels observed in control myocytes not treated with PE. This Cn-mediated increase in Ito,f back to control levels occurred despite the fact that Cn increased Kv4.2 protein expression by 2-fold (and Kv4.2 mRNA transcription by ~5-fold) above the control in PE-treated myocytes. Returning the Ito,f to control levels in PE-treated myocytes is not unexpected because Cn had no effect on expression of Kv4.3 or KChIP2, which are both required for co-assembly with Kv4.2 as heterotetramers (1) to form functional Ito,f channels (35). Additional studies will clearly be required to determine the cellular mechanisms underlying the reductions in Kv4.2, Kv4.3, and KChIP2 expression induced by {alpha}1AR stimulation, which activates numerous hypertrophic signaling pathways (44).

The enhanced Ito,f by Cn observed in our study may represent an important feedback pathway operating under conditions of cardiac hypertrophy and heart disease. Specifically, as already mentioned, many hypertrophic stimuli, including {alpha}1AR stimulation and heart disease, reduce Ito,f as well as Kv4.2, Kv4.3, and KChIP2 expression (8, 45, 46). These conditions have also been linked to Cn activation (8, 45, 47), which is expected from our results to limit the extent of Ito,f down-regulation. Preventing excessive Ito,f reductions may be of critical importance because changes in Ito,f can lead to altered electrical and contractile properties of the myocardium, which may contribute to impaired pump function and arrhythmias (6, 48-51). For example, we (48, 52) and others (53) have shown previously that not only is Ito,f an important determinant of action potential profile changes occurring in heart disease and regional differences in action potential profile, but Ito,f is also a major regulator of the amplitude and kinetics of cardiac contraction in rodents and larger mammals by modulating Ca2+ entry via L-type Ca2+ channels. Thus, Cn activation not only induces hypertrophy, but also modulates the extent of the electrical and contractile changes induced as a result of Ito,f reduction caused by hypertrophic stimuli. In addition, a recent study found that increased Ito,f by adenoviral overexpression of Kv4.3 can prevent hypertrophy (45), whereas increased Ito,f in NRVMs treated with PE prevents both myocyte hypertrophy and Cn activation (8), suggesting that, at least in these models, increased Ito,f by Cn could limit the degree of hypertrophy.

In conclusion, we have found that Cn/NFAT potently increases Kv4.2 transcription and Ito,f levels in rodent myocardium. This regulation provides a potentially useful mechanism for modulating the cellular, electrical, and contractile responses of the myocardium to cardiac hypertrophy and heart disease. The relevance of Cn-mediated regulation of Kv4.2 to larger mammalian species such as humans and dogs is uncertain because, in these species, Ito,f is encoded by Kv4.3 and KChIP2 subunits. Additional studies will be required to completely define the role of Cn in the regulation of Ito,f in non-rodents.


   FOOTNOTES
 
* This work was supported in part by the Canadian Institutes of Health Research (to P. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada, the Canadian Institutes of Health Research Tailored Advanced Collaborative Training in Cardiovascular Science Program, and the Faculty of Medicine at the University of Toronto. Back

2 Established Investigator of the American Heart Association. Back

3 To whom correspondence should be addressed: Heart and Stroke/Richard Lewar Centre of Excellence, Fitzgerald Bldg., 150 College St., Toronto, Ontario M5S 3E2, Canada. Tel.: 416-949-8112; Fax: 416-949-8380; E-mail: p.backx{at}utoronto.ca.

4 The abbreviations used are: Ito,f, fast transient outward K+ current; {alpha}1AR, {alpha}1-adrenoreceptor; Cn, calcineurin; NFAT, nuclear factor of activated T-cells; NRVMs, neonatal rat ventricular myocytes; PE, phenylephrine; CAIN, calcineurin inhibitory peptide; GFP, green fluorescent protein; Gmax, maximal chord conductance; V1/2, voltage for half-maximal activation of Ito,f; Gslope, slope conductance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Isus, sustained current; pF, picofarad. Back

5 N. Gong and P. H. Backx, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank D. Zhao and Z. Kassiri for amplification of the recombinant adenoviruses. We acknowledge the kind gifts of Kv4.2 promoters from Dr. K. Takimoto, Kv4.3 promoters from Dr. E. S. Levitan, the 3xNFAT-luc vector from Dr. A. Rao, and VIVIT adenoviruses from Dr. S. D. Kraner.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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