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(Circulation Research. 2006;98:1306.)
© 2006 American Heart Association, Inc.

Cellular Biology

Differential Calcineurin/NFATc3 Activity Contributes to the I_to Transmural Gradient in the Mouse Heart

Charles F. Rossow, Keith W. Dilly, Luis F. Santana

From the Department of Physiology and Biophysics, University of Washington, Seattle.

Correspondence to L.F. Santana, University of Washington, Department of Physiology and Biophysics, Box 357290, Seattle, WA 98118. E-mail santana{at}u.washington.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kv4 channels are differentially expressed across the mouse left ventricular free wall. Accordingly, the transient outward K⁺ current (I_to), which is produced by Kv4 channels, is greater in left ventricular epicardial (EPI) than in endocardial (ENDO) cells. However, the mechanisms underlying heterogeneous Kv4 expression in the heart are unclear. Here, we tested the hypothesis that differential [Ca²⁺]_i and calcineurin/NFATc3 signaling in EPI and ENDO cells contributes to the gradient of I_to function in the mouse left ventricle. In support of this hypothesis, we found that [Ca²⁺]_i, calcineurin, and NFAT activity were greater in ENDO than in EPI myocytes. However, the amplitude of I_to was the same in ENDO and EPI cells when [Ca²⁺]_i, calcineurin, and NFAT activity were equalized. Consistent with this, we observed complete loss of I_to and Kv4 heterogeneity in NFATc3-null mice. Interestingly, Kv4.3, Kv4.2, and KChIP2 genes had different apparent thresholds for NFATc3-dependent suppression and were ordered as Kv4.3KChIP2>Kv4.2. Based on these data, we conclude that calcineurin and NFATc3 constitute a Ca²⁺-driven signaling module that contributes to the nonuniform distribution of Kv4 expression, and hence I_to function, in the mouse left ventricle.

Key Words: signal transduction • arrhythmias • voltage-gated potassium currents • calcium • gene regulation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The transient outward K⁺ current (I_to) contributes to the early repolarization phase of the ventricular action potential. In mice, a heterotetramer of pore-forming Kv4.2 and Kv4.3 subunits underlie I_to.¹ In human and canine, which do not express Kv4.2, I_to is produced by Kv4.3 subunits. A distinctive feature of the mammalian heart is that I_to function varies throughout the myocardium.^2,3 Indeed, there is a striking difference in the amplitude of I_to between left ventricular epicardial (EPI) than in endocardial (ENDO) cells,^2,3 with I_to being larger in EPI than in ENDO myocytes.^2,4,5 This transmural gradient of I_to function is important for normal ventricular repolarization.^4,6,7

Previous work examining the mechanisms underlying regional differences in I_to function have focused on determining the expression patterns of Kv4 and the accessory KChIP2 subunit in different regions of the heart. KChIP2 acts as a chaperone that increases Kv4 surface expression and hence increases I_to.^4,8 In addition to this, KChIP2 has also been shown to modulate the rate of inactivation and voltage dependence of Kv4 channels.^9,10 In humans and canine, differential Kv4.3 and/or KChIP2 expression^3,11,12 is thought to control regional differences in I_to function across the ventricular wall. In contrast, in the mouse ventricle, where a transmural gradient in KChIP2 and Kv4.3 is absent,¹³ differential Kv4.2 expression presumably underlies heterogeneous I_to function.^1,5,13 At present, however, the mechanisms underlying regional differences in Kv4 expression are unclear.

We recently demonstrated that activation of the transcription factor NFATc3 by the Ca²⁺-dependent phosphatase calcineurin transduces variations in [Ca²⁺]_i into changes in I_to function in ventricular myocytes.¹⁴ NFATc3 decreased I_to by reducing Kv4 expression. Here, we tested the hypothesis that differential [Ca²⁺]_i and calcineurin/NFATc3 signaling in EPI and ENDO cells contributes to the gradient of I_to function in the mouse left ventricle.

We found that [Ca²⁺]_i and calcineurin/NFATc3 activity is higher in ENDO than in EPI myocytes, resulting in lower Kv4 expression and I_to function in ENDO cells. Importantly, we show that NFATc3 expression was necessary for differential I_to function across the left ventricular wall. Furthermore, our data indicate that Kv4.2, Kv4.3, and KChIP2 genes vary with regard to the level of calcineurin/NFAT activity required for their downregulation. We conclude that differential patterns of [Ca²⁺]_i, calcineurin, and NFATc3 signaling contribute to regional differences in I_to function in the mouse myocardium.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocytes were obtained from the left ventricle ENDO and EPI of wild-type (balbc), NFAT-null (NFAT^–/–; kindly provided by Dr Laurie Glimcher), and NFAT-luc (kindly provided by Dr Jeffrey Molkentin), as previously described.⁵ Electrophysiological signals were recorded using an Axopatch 200B. For [Ca²⁺]_i measurements cells, were loaded with the acetomethylester version of the fluorescent Ca²⁺ indicator fluo-4–acetoxymethyl ester. RT-PCR and Western blot analyses were performed as described elsewhere.¹⁴ Calcineurin activity was quantified using a commercially available kit (Promega). Data are presented as mean±SEM.

An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Higher [Ca²⁺]_i Underlies Increased Calcineurin Activity in the ENDO Than in the EPI
Figure 1A shows field-stimulated (1 Hz) whole-cell [Ca²⁺]_i transients recorded from representative ENDO and EPI cells. We found that peak systolic [Ca²⁺]_i was higher in ENDO cells (942±127 nmol/L, n=6) than in EPI cells (491±79 nmol/L, n=6; P<0.05; Figure 1B). Interestingly, ENDO cells (279±44 nmol/L, n=6) also had higher diastolic [Ca²⁺]_i than EPI cells (154±36 nmol/L, n=6; P<0.05; Figure 1C). Higher diastolic and systolic [Ca²⁺]_i levels were observed in ENDO versus EPI over a broad range of stimulation frequencies (0.25 to 3 Hz; data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 1. Diastolic and systolic [Ca2+]i is higher in ENDO than in EPI cells. A, Representative [Ca2+]i transients from ENDO and EPI cells. B and C, Bar plots show the mean±SEM of the peak systolic (B) and diastolic (C) [Ca2+]i transient in EPI and ENDO cells.

We investigated the possibility that the differences in [Ca²⁺]_i between EPI and ENDO cells described above could lead to regional variations in the activity of calcineurin/NFAT signaling. We focused on this signaling pathway for the following reasons. First, the generally accepted view is that calcineurin and NFAT activity is higher in cells with elevated [Ca²⁺]_i.^14,15,16 Second, NFATc3 activates the hypertrophic gene program, which includes downregulation of Kv4 channel expression.^14,17 Because ENDO cells have higher [Ca²⁺]_i and express less Kv4 than EPI cells, we hypothesized that calcineurin/NFAT activity is higher in ENDO than in EPI cells.

To test this hypothesis, calcineurin activity in EPI and ENDO cells was examined (Figure 2A). To ensure that calcineurin activity was assessed in these cells under near physiological conditions, beating hearts were rapidly removed from the chest cavity and placed in ice-cold saline solution in which the EPI and ENDO were quickly separated and snap-frozen in liquid nitrogen. Using this approach, we found that calcineurin activity was significantly higher in ENDO than in EPI (n=5; P<0.05).

View larger version (23K): [in this window] [in a new window]   Figure 2. Differences in calcineurin activity between ENDO and EPI. A, Bar plot of the mean±SEM of calcineurin activity in ENDO and EPI. B, Western blot of calcineurin A ß protein in ENDO and EPI. The bar plot shows the amount of calcineurin A ß in ENDO (relative to EPI). C, Time course of [Ca2+]i during steady-state stimulation (1 Hz) and after cessation of pacing in typical ENDO (red) and EPI (black) cells. The inset shows a bar plot of the mean±SEM of the normalized (to mean EPI calcineurin activity under control conditions) calcineurin activity in quiescent ENDO and EPI cells. Resting [Ca2+]i in these cells was 162±20 nmol/L.

Differences in calcineurin activity between ENDO and EPI could be attributable to differences in the expression of this phosphatase. Thus, we used Western blot analysis to determine protein levels of the ß subunit of calcineurin A—the predominant calcineurin isoform in heart¹⁸—in EPI and ENDO. As shown in Figure 2B, calcineurin A ß protein is expressed to a similar extent in EPI and ENDO (n=5 hearts, P>0.05). Thus, increased calcineurin A ß expression does not account for the greater activity of this phosphatase in ENDO than in EPI.

We investigated whether differences in calcineurin activity between EPI and ENDO myocytes were caused by differences in [Ca²⁺]_i between these cells. As noted above, during stimulation, systolic and diastolic [Ca²⁺]_i was higher in ENDO than in EPI cells. However, after pacing was discontinued, diastolic [Ca²⁺]_i was similar in ENDO and EPI cells (n=18, P>0.05; Figure 2C). Indeed, the average diastolic [Ca²⁺]_i in quiescent EPI and ENDO cells was 162±20 nmol/L. Calcineurin activity was measured in EPI and ENDO cells that had remained quiescent for a minimum of 2 hours. Consistent with our hypothesis, we found that under these conditions (ie, resting [Ca²⁺]_i160 nmol/L), calcineurin activity was similar in EPI and ENDO cells. Taken together, these data suggest that transmural variations in [Ca²⁺]_i underlie the differences in calcineurin activity in the EPI and ENDO.

Higher NFATc3 Activity in the ENDO Than in the EPI
We investigated whether the differences in calcineurin activity between the EPI and ENDO described above translated into regional differences in NFAT activity. In these experiments, we used a transgenic NFAT reporter mouse (NFAT-luc), in which luciferase expression is controlled by multiple NFAT binding sites.¹⁹ We measured luciferase transcript levels in NFAT-luc ENDO and EPI tissue. As a control, the expression level of ß-actin mRNA was also measured in these tissues. Consistent with our hypothesis, we found that luciferase, but not ß-actin, transcript expression was 2-fold higher (n=5, P<0.05) in ENDO than in EPI (Figure 3A). These data corroborate our calcineurin activity data and suggest that NFAT activity is higher in ENDO than in EPI.

View larger version (20K): [in this window] [in a new window]   Figure 3. Higher NFAT activity in ENDO than in EPI. A, RT-PCR analysis of luciferase transcript in ENDO and EPI tissue isolated from NFAT-luc transgenic mice. The images show transcript amplification products for luciferase (510 bp) and ß-actin (496 bp). The graph plots the mean±SEM of the relative (EPI/ENDO) band intensity. B, Western blot analysis of NFATc3 and ß-actin protein in ENDO and EPI. The bar plot shows the amount of NFATc3 and ß-actin protein in EPI relative to ENDO cells. C, Western blot gels of total (GSK3ß, top) and phosphorylated (p-GSK3ß, bottom) GSK3ß protein in ENDO and EPI. Bar plot of the mean±SEM of the relative (EPI/ENDO) GSK3ß and p-GSK3ß.

Next, we measured NFATc3 protein in EPI and ENDO cells. We focused on NFATc3 because we,¹⁴ and others,²⁰ have shown that NFATc3 is essential in the pathway leading to cardiac hypertrophy and Kv4 downregulation following calcineurin activation. Analogous to calcineurin, we found that NFATc3 (and ß-actin) protein levels were similar in EPI and ENDO tissue (Figure 3B), indicating that increased NFATc3 expression does not account for greater NFAT activity in the ENDO than in EPI. Our data are consistent with the view that higher calcineurin activity results in greater NFAT activity in the ENDO.

Increased NFAT activity could be caused by increased nuclear import and/or decreased export of this transcription factor in ENDO than in EPI cells. Nuclear export of NFAT is promoted by the activity of glycogen synthase kinase-3 ß (GSK3ß).²¹ On phosphorylation, GSK3ß (p-GSK3ß) is activated, thus phosphorylating conserved serines within the NFAT N terminus that are required for its nuclear export. Thus, we examined GSK3ß activity in ENDO and EPI (Figure 3C). We found that the levels of total GSK3ß (ie, inactive kinase) and p-GSK3ß (ie, active kinase) protein were similar in ENDO and in EPI cells (n=5, P>0.05), indicating that GSK3ß activity is similar in ENDO and EPI cells. Together with our calcineurin activity data described above, this suggests that increased nuclear import of NFAT, not decreased export by p-GSK3ß, underlies higher NFAT activity in ENDO than in EPI.

NFATc3 Is Required for Establishing the I_to Gradient Across the Left Ventricular Wall
Increased calcineurin/NFATc3 activity downregulates Kv4 channel expression.¹⁴ Thus, we hypothesized that NFATc3 activity is required for the transmural I_to gradient. A testable prediction of this hypothesis is that mice lacking NFATc3 expression (NFATc3^–/–) would, at a minimum, have a less pronounced gradient of I_to function across the ventricular wall.

I_to currents were recorded in EPI and ENDO cells isolated from wild-type (WT) and NFATc3^–/– hearts (Figure 4A). I_to was isolated from other voltage-gated K⁺ currents in these cells by pharmacological means, as previously described.²² I_to was evoked by 1.2-second step depolarizations from a holding potential of –90 mV to voltages ranging from –60 to +40 mV and was defined as the difference between the peak and the sustained current measured at the end of the pulse.

View larger version (21K): [in this window] [in a new window]   Figure 4. NFATc3 expression is required for the transmural Ito gradient across the left ventricular free wall. A, Representative Ito records from WT (left) and NFATc3–/– (right) EPI (black) and ENDO (red) cells during depolarizations from –90 to +40 mV. B, Current–voltage relationships of Ito in WT and NFATc3–/– EPI and ENDO cells.

To begin, we recorded I_to in WT ENDO and EPI myocytes. As previously reported,⁵ I_to was larger in WT EPI than in ENDO cells at most voltages examined (Figure 4A and 4B). Surprisingly, we found that I_to was similar in EPI and ENDO cells from NFATc3^–/– hearts. Note that equalization of I_to amplitudes between ENDO and EPI cells from NFATc3^–/– hearts was produced by increased I_to function in ENDO cells as opposed to decreased I_to in EPI cells. These data are consistent with our observation that basal NFATc3 activity is higher in WT ENDO than in EPI cells and suggest, for the first time, that NFATc3 is necessary in establishing the gradient of I_to across the left ventricular wall.

We investigated the molecular mechanisms by which NFATc3 regulates I_to function. In mouse ventricular myocytes, I_to is produced by pore-forming Kv4.2 and Kv4.3 -subunits and accessory KChIP2 proteins.^1,4 RT-PCR was used to measure Kv4.2, Kv4.3, and KChIP2 transcripts in WT ENDO and EPI cells. As previously reported,¹³ WT EPI cells expressed more Kv4.2 transcript than ENDO (P<0.05, n=5), and Kv4.3 and KChIP2 transcript levels were similar in WT ENDO and EPI cells (P>0.05, n=5; data not shown). Together, these data are consistent with the view that, in mice,^1,5,13 the transmural gradient of I_to is attributable to differential expression of the Kv4.2 subunit.

Next, we examined Kv4.2 and Kv4.3 protein expression in EPI and ENDO cells from WT and NFATc3^–/– hearts (Figure 5). Similar to our transcript data above, and as reported by others,¹³ we found that WT EPI cells expressed more Kv4.2 protein (Figure 5A) than ENDO cells; Kv4.3 was similar between ENDO and EPI cells (Figure 5B). Importantly, and in agreement with our electrophysiological data, Kv4.3 and Kv4.2 expression was not different between NFATc3^–/– EPI and ENDO cells (P>0.05; Figure 5A and 5B). ß-Actin expression was similar (P>0.05) in WT and NFATc3^–/– EPI and ENDO cells (Figure 5C). It is important to note that, consistent with our electrophysiological data, Kv4.3 expression (normalized to ß-actin) was similar in WT and NFATc3^–/– ENDO and EPI cells (P>0.05). These findings suggest that differential expression of I_to between EPI and ENDO results from NFATc3-dependent regional differences in Kv4.2 expression.

View larger version (27K): [in this window] [in a new window]   Figure 5. NFATc3 regulates Kv4 expression across the left ventricular free wall. A through C, Western blot analysis of Kv4.2 (A), Kv4.3 (B), and ß-actin protein (C) in WT and NFATc3–/– EPI and ENDO. The bar plots show the relative level of these proteins in ENDO (compared with EPI). D, Bar plots of the normalized IRX5 (to ß-actin) transcript in WT and NFATc3–/– EPI and ENDO cells.

A recent study suggested that differential expression of the transcription factor IRX5 contributes to heterogeneous Kv4.2 expression across the mouse left ventricular wall.¹³ These findings raised the intriguing possibility that loss of NFATc3 could have lead to loss of the Kv4.2 and I_to gradient indirectly through changes in IRX5 expression. To test this hypothesis, we measured IRX5 transcript in WT and NFATc3^–/– ENDO and EPI myocytes (Figure 5D). Consistent with Costantini et al,¹³ we found IRX5 expression was 6-fold higher in WT ENDO than in EPI cells (n=6 amplifications from 3 hearts; P<0.05). Like in WT hearts, IRX5 transcript expression was 7-fold higher in NFATc3^–/– ENDO than in EPI cells (n=6 amplifications from 3 hearts; P<0.05). Together with our electrophysiological data, these findings suggest that in the absence of NFATc3, differential IRX5 expression in ENDO and EPI cells is not sufficient to produce heterogeneous Kv4.2 expression and I_to function across the left ventricular wall.

EPI and ENDO Cells With Matching Calcineurin Activities Have Similar I_to Amplitudes
After having established that greater basal NFATc3 activity decreased Kv4.2 expression and I_to function in ENDO cells, we investigated the relationship between calcineurin activity and I_to in EPI and ENDO cells. If calcineurin negatively regulated I_to (via NFATc3), as we have proposed, then a sustained increase in calcineurin activity in EPI cells should reduce the amplitude of I_to in these cells. Conversely, I_to should increase in ENDO cells in response to a sustained decrease in calcineurin activity.

We investigated the effects of matching calcineurin activity in ENDO and EPI cells on I_to. In these experiments, we took advantage of our observation that diastolic [Ca²⁺]_i in ENDO cells decreased from 275 nmol/L during stimulation to 160 nmol/L during rest (see Figure 2C and above). Note that at this [Ca²⁺]_i, calcineurin activity in ENDO cells is reduced to the same level as observed in EPI cells (see Figure 2C, inset, and above). We recorded I_to in ENDO and EPI cells that had been cultured for 24 hours under these conditions (ie, no pacing). For comparison, I_to records from freshly dissociated EPI and ENDO cells are included in these plots (Figure 6A). We found that after 24 hours of decreased [Ca²⁺]_i and calcineurin activity, the amplitude of I_to (at +40 mV) in ENDO cells doubled from 20 pA/pF (in acutely dissociated ENDO cells) to 40 pA/pF (Figure 6A and 6B). Importantly, the amplitude of I_to in EPI (acutely dissociated and cultured cells) and ENDO cells with matching [Ca²⁺]_i was not different.

View larger version (21K): [in this window] [in a new window]   Figure 6. Modulation of Ito expression in EPI and ENDO cells by changes in calcineurin activity. A, Representative traces of Ito in freshly dispersed (data from Figure 1) and cultured ENDO and EPI myocytes. ENDO and EPI cells were cultured for 24 hours under conditions in which external Ca2+ was 2 or 5 mmol/L. ENDO and EPI cells were also cultured in the presence of 5 mmol/L external Ca2+ and 10 µmol/L of CiP. Parallel experiments indicated that resting [Ca2+]i was 160 nmol/L and 270 nmol/L with 2 and 5 mmol/L external Ca2+, respectively. B, Bar plot of mean±SEM of Ito at +40 mV from freshly dispersed ENDO and EPI myocytes (data from Figure 1) and ENDO and EPI myocytes cultured under indicated culture conditions. The black dotted lines in A and B indicate the amplitude of Ito in control, freshly dispersed EPI cells.

Next, we performed the converse experiment: EPI and ENDO cells were cultured with an external solution containing 5 mmol/L Ca²⁺. At this external [Ca²⁺], resting [Ca²⁺]_i in EPI and ENDO cells was 270±18 nmol/L (n=12), which is similar (P>0.05) to the observed diastolic [Ca²⁺]_i in paced ENDO cells (279±44 nmol/L; Figure 1). At this relatively high resting [Ca²⁺]_i, calcineurin activity in ENDO and EPI cells is expected to be higher¹⁵ than in cells with the lower resting [Ca²⁺]_i of 160 nmol/L and similar to freshly dissociated ENDO cells (see Figure 2A and above). To increase cell viability, tetracaine (50 µmol/L) was included in the culture medium to suppress spontaneous Ca²⁺ release from the sarcoplasmic reticulum.

We found that increasing diastolic [Ca²⁺]_i to 275 nmol/L decreased the amplitude of I_to in EPI cells to a level similar to acutely dissociated ENDO cells (P>0.05; Figure 6A and 6B). Note that under these experimental conditions, I_to was similar in cultured and acutely dissociated ENDO cells. These data indicate that increasing [Ca²⁺]_i reduces I_to in EPI cells. Furthermore, our data indicate that the higher [Ca²⁺]_i in ENDO cells in vivo contributes to the smaller I_to observed in these cells.

We investigated whether the observed reduction of I_to in ENDO and EPI cells following increased [Ca²⁺]_i depends on activation of calcineurin. To test this hypothesis, ENDO and EPI cells were cultured in high Ca²⁺ medium (5 mmol/L) containing the specific calcineurin autoinhibitory peptide (CiP). We found that in the presence of the calcineurin CiP (10 µmol/L), high [Ca²⁺]_i did not change the amplitude of I_to in either EPI and ENDO cells (n=7, P>0.05; Figure 6A and 6B). This suggests that calcineurin activity is necessary for high [Ca²⁺]_i-mediated downregulation of I_to. Taken together, our data suggest that the amplitude of I_to depends on the level of diastolic [Ca²⁺]_i and calcineurin activity in left ventricular myocytes. Accordingly, we propose that the transmural gradient of I_to function results from regional differences in [Ca²⁺]_i and calcineurin (and thus NFATc3) activity.

Downregulation of Kv4.3, Kv4.2, and KChIP2 mRNA at Different [Ca²⁺]_i and Calcineurin/NFAT Activities
Analysis of the Kv4.2 gene revealed putative NFAT binding sites in its promoter region.¹⁴ However, putative NFATc3 binding sites can also be found in the Kv4.3¹⁴ and KChIP2 (NM145704: –293, –415, –812, –1034) genes, which together with Kv4.2, underlie I_to in the mouse ventricle. This raises an important question: If these 3 genes have putative NFAT binding sites, why is Kv4.2 the only gene downregulated in ENDO cells? An intriguing possibility is that Kv4.2, Kv4.3, and KChIP2 have different thresholds for NFAT-dependent suppression.

To test this hypothesis, we examined Kv4.3, Kv4.2, and KChIP2 transcripts in ventricular myocytes cultured for 24 hours with 2, 5, and 10 mmol/L external Ca²⁺. In parallel experiments, we used ventricular myocytes infected with an adenoviral vector expressing NFATc3 fused to the monomeric red fluorescent protein 1 (NFATc3-mRFP1; see online data supplement for details about the construction of this vector and analysis of these data) to monitor the levels nuclear NFATc3-mRFP1 at these Ca²⁺ concentrations. Similarly, steady-state resting [Ca²⁺]_i was measured in cells exposed to 2, 5, and 10 mmol/L external [Ca²⁺]_i for 24 hours.

As shown in Figure 7A, [Ca²⁺]_i and nuclear NFATc3-mRFP1 levels increased as external Ca²⁺ was increased from 2 to 5 and 10 mmol/L. Note, however, that in the presence of CiP (10 µmol/L), increasing external Ca²⁺ from 2 to 10 mmol/L did not evoke an increase in nuclear NFATc3-mRFP1 (P>0.05). This suggests that the increase in nuclear NFATc3-mRFP1 evoked by 10 mmol/L Ca²⁺ requires calcineurin activity.

View larger version (23K): [in this window] [in a new window]   Figure 7. Kv4 and KChip2 transcripts are downregulated at different external Ca2+, [Ca2+]i, and NFAT activities in mouse ventricular myocytes. A, Plot of the resting [Ca2+]i and relative nuclear NFATc3-mRFP1 translocation (normalized to 2 mmol/L) as a function of [Ca2+]o. The relative nuclear NFATc3-mRFP1 translocation at [Ca2+]o=10 mmol/L in the presence of CiP (10 µmol/L) is also shown. B and C, Plots of the relative Kv4.2, Kv4.3, and KChIP2 transcript (normalized to 2 mmol/L) in ventricular myocytes cultured at varied [Ca2+]o under control conditions (B) and (C) in the presence of CiP (10 µmol/L).

We found that increasing [Ca²⁺]_o from 2 to 5 mmol/L, which caused a 1.6±0.1-fold increase in nuclear NFATc3-mRFP1 (n=5, P<0.05), decreased Kv4.2 (n=9, P<0.05) by 60% but not KChIP2 or Kv4.3 transcript (n=9, P>0.05; Figure 7A and 7B). Interestingly, a further increase in [Ca²⁺]_o to 10 mmol/L, which elicited a 3.1±0.4-fold (n=5, P<0.05; Figure 7A) increase in nuclear NFATc3-mRFP1, decreased Kv4.2, Kv4.3, and KChIP2 by the same extent (P>0.05; Figure 7B). It is important to note that in the presence of CiP (Figure 7C) or in cells loaded with the Ca²⁺ buffer BAPTA–acetoxymethyl ester (5 µmol/L; data not shown), increasing external Ca²⁺ from 2 to 10 mmol/L did not change (P>0.05) Kv4 or KChIP2 expression, indicating that downregulation of these genes in elevated external Ca²⁺ requires Ca²⁺-dependent activation of calcineurin.

For comparison, we also measured Kv2.1 and Kv1.5 transcript levels in myocytes cultured in 2, 5, and 10 mmol/L Ca²⁺ (see online data supplement, Figure I). Kv1.5 and Kv2.1 genes, which underlie I_K,slow (a voltage-gated, rapidly activating, and slowly inactivating current) in mouse ventricular myocytes,^23,24 have putative NFAT binding sites in their promoters.¹⁴ A previous study¹³ has shown that I_K,slow and Kv1.5 expression is similar in mouse ENDO and EPI cells. Consistent with this, we detected similar Kv2.1 transcript levels in ENDO and EPI cells (P<0.05; data not shown).

We found that, like Kv4.3 and KChIP2, increasing external Ca²⁺ from 2 to 5 mmol/L did not decrease Kv1.5 or Kv2.1 transcript expression. At 10 mmol/L Ca²⁺, Kv2.1 transcript was decreased by 80% (P<0.05; compared with 2 mmol/L).Kv1.5 transcript was similar at all [Ca²⁺]_o examined. Together, these data suggest that Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChiP2 genes vary with regard to the level of [Ca²⁺]_i and calcineurin/NFAT activity required for their downregulation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We provide evidence that regional differences in calcineurin/NFATc3 signaling contribute to heterogeneous I_to function across the mouse left ventricular free wall. Our data suggest that higher [Ca²⁺]_i in ENDO than in EPI cells underlies higher calcineurin/NFATc3 signaling activity in ENDO than in EPI cells. We recently examined the mechanisms underlying differential [Ca²⁺]_i across the mouse left ventricular wall.²⁵ The Ca²⁺ current was similar in mouse EPI and ENDO cells. However, we found that the longer action potential and lower Na⁺/Ca²⁺ exchanger function of mouse ENDO cells results in higher Ca²⁺ influx and lower Ca²⁺ extrusion than in EPI cells, resulting in higher diastolic and systolic [Ca²⁺]_i in ENDO than in EPI cells during pacing.

The data presented here indicate that these regional differences in [Ca²⁺]_i could indirectly regulate I_to function through activation of calcineurin/NFATc3 signaling. Although the nature of the [Ca²⁺]_i signal responsible for the activation of the calcineurin/NFAT pathway in ventricular myocytes is unclear, our data suggest that higher diastolic [Ca²⁺]_i is sufficient to reduce I_to in ENDO cells. We,¹⁴ and others,²⁶ have demonstrated that increased calcineurin activity is transduced into a reduction of I_to through activation of NFATc3. The absence of a transmural I_to gradient in NFATc3^–/– ventricles strongly supports this hypothesis and indicates the necessity of this transcription factor for regional variations in I_to. Careful analysis of our NFATc3^–/– data shows that I_to homogeneity in these hearts resulted from increased I_to in ENDO cells; there were no differences in I_to in EPI cells from WT and NFATc3^–/– hearts. When placed in context with our calcineurin and NFAT activity data, these findings provide compelling support to the view that basal calcineurin/NFATc3 activity is higher in the ENDO than in the EPI.

Our study suggests a well-defined molecular mechanism underlying lower Kv4.2 expression in ENDO cells: higher [Ca²⁺]_i in ENDO cells activates calcineurin, which then dephosphorylates and hence activates NFATc3. Once activated, NFATc3 translocates into the nuclei, where it alters gene expression. NFAT is exported from the nuclei by GSK3ß-dependent phosphorylation. Our data suggest that increased import, not decreased GSK3ß-mediated export, underlies higher NFAT activity in ENDO than in EPI cells.

An important observation in this study is that Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChIP2 genes, all of which have putative NFAT binding sites in their promoters, have different thresholds for NFAT-dependent suppression. Our data indicate that at low levels of NFAT activity (ie, similar to those at 2 mmol/L external Ca²⁺), expression of Kv1.5, Kv2.1, Kv.4.2, Kv4.3, and KChIP2 is high. Increasing NFAT activity by 1.6-fold downregulated Kv4.2, but not Kv1.5, Kv2.1, Kv4.3, or KChIP2, expression in mouse ventricular myocytes; much higher increases (presumably 3-fold) in NFAT activity are required for downregulation (60%) of all of these genes. Consistent with these data, we recently reported¹⁴ that expression of a constitutively active NFAT or a 4-fold increase in NFAT activity after myocardial infarction activity decreases Kv1.5, Kv2.1, Kv4.2, and Kv4.3 in ventricular myocytes by 60% (KChIP2 was not measured in this study).

Based on these findings, we have formulated a model in which the level of Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChiP2 expression depends on the relative level calcineurin/NFAT activity throughout the mouse heart. In this model, low levels of NFAT activity similar to those observed in mouse EPI and in ventricular myocytes cultured in 2 mmol/L Ca²⁺ fall below the threshold for NFAT-induced downregulation, which ensures high Kv1.5 Kv2.1, Kv.4.2, Kv4.3, and KChIP2 expression and hence I_K,slow and I_to function in these cells. We propose that in ENDO, which has nearly 2-fold higher NFAT activity than EPI (ie, similar to cells cultured in 5 mmol/L Ca²⁺), the activity of this transcription factor reaches a threshold for downregulation of Kv4.2, but not Kv1.5, Kv2.1, Kv4.3, and KChIP2, in these cells. Because Kv1.5¹³ and Kv2.1 expression is similar in mouse EPI and ENDO, I_K,slow is similar in these cells.¹³ Our data suggesting that Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChIP2 expression and I_to function depend on the relative activity of calcineurin/NFAT signaling in ventricular myocytes strongly support this model.

The transmural I_to gradient in canine and human hearts is produced by differential Kv4.3¹² and/or KChIP2^3,11 expression between ENDO and EPI cells. Note that, like in mouse, diastolic and systolic [Ca²⁺]_i is higher in canine ENDO than in EPI.²⁷ Thus, it is intriguing to speculate that differential calcineurin/NFAT activity across the left ventricular wall underlies KChIP2 and Kv4.3 expression and thus I_to function in these cells in larger mammals. Experiments need to be performed to establish the relationship between calcineurin/NFAT signaling and KChIP2 and Kv4.3 expression in these species.

As noted above, a recent study¹³ reported complete loss of the I_to gradient in hearts lacking expression of the transcription factor IRX5. On the basis of these findings and the observation that IRX5 was expressed to a larger extent in ENDO than in EPI cells, it was suggested that differential expression of this transcription factor suppressed Kv4.2 expression and hence reduced I_to to a larger extent in ENDO than in EPI cells. However, we found that the IRX5 gradient persists in NFATc3^–/– hearts, which have homogeneously distributed I_to function. This indicates that although IRX5 is necessary for heterogeneous expression of Kv4.2, it is by itself not sufficient. Indeed, because Costantini et al¹³ did not measure calcineurin/NFAT activity in IRX5^–/– EPI and ENDO, the possibility that loss of the transmural I_to gradient is attributable to changes in calcineurin/NFAT activity in these cells cannot be completely ruled out. Nonetheless, their study¹³ and ours clearly indicate that NFATc3 and IRX5 expression are required for differential I_to function across the left ventricular free wall. It is important to note, however, that data presented here and in a previous study¹⁴ suggest that activation of NFAT in IRX5-expressing myocytes is sufficient to downregulate Kv4.2 expression and I_to function in these cells. Future experiments should investigate the relationship among IRX5, NFATc3, and Kv4 expression in ENDO and EPI cells.

A recent study²⁸ suggested that activation of mitogen-activated protein kinase (MAPK) pathways decrease I_to function in ventricular myocytes by downregulating KChIP2 expression during the development of hypertrophy and heart failure. Activation of MAPK signaling presumably downregulates KChIP2 transcript expression through the activation of JNK1 and MEK1-ERK. Note, however, that KChIP2 transcript does not vary across the left ventricular wall of rodents.⁵ Thus, although activation of MAPK pathways may lead to downregulation of KChIP2 and consequently to I_to function during pathological conditions, they do not appear to contribute to differential I_to function across the left ventricular wall, at least in rodents, during physiological conditions.

In summary, our data clearly indicate that regional variations in [Ca²⁺]_i and calcineurin/NFATc3 activity in the left ventricular free wall contribute to the nonuniform distribution of I_to function in the mouse ventricle. Furthermore, our data establishes the importance of calcineurin/NFATc3 signaling in modulating ventricular excitability under physiological conditions.

	Acknowledgments

 
This work was supported by NIH and American Heart Association grants. We thank Drs Gregory Amberg, Manuel Navedo, Madeline Nieves, and Carmen Ufret-Vincenty for reading the manuscript. We also thank Jennifer Cabarrus for technical assistance.

	Footnotes

 
Original received October 25, 2005; resubmission received February 28, 2006; revised resubmission received March 20, 2006; accepted March 30, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Guo W, Li H, Aimond F, Johns DC, Rhodes KJ, Trimmer JS, Nerbonne JM. Role of heteromultimers in the generation of myocardial transient outward K⁺ currents. Circ Res. 2002; 90: 586–593.[Abstract/Free Full Text]
2. Clark RB, Bouchard RA, Salinas-Stefanon E, Sanchez-Chapula J, Giles WR. Heterogeneity of action potential waveforms and potassium currents in rat ventricle. Cardiovasc Res. 1993; 27: 1795–1799.[Abstract/Free Full Text]
3. Rosati B, Pan Z, Lypen S, Wang HS, Cohen I, Dixon JE, McKinnon D. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J Physiol. 2001; 533: 119–125.[Abstract/Free Full Text]
4. Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I_to and confers susceptibility to ventricular tachycardia. Cell. 2001; 107: 801–813.[CrossRef][Medline] [Order article via Infotrieve]
5. Brunet S, Aimond F, Guo W, Li H, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K⁺ currents in adult mouse ventricles. J Physiol. 2004; 559: 103–120.[Abstract/Free Full Text]
6. Barry DM, Xu H, Schuessler RB, Nerbonne JM. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit. Circ Res. 1998; 83: 560–567.[Abstract/Free Full Text]
7. Guo W, Li H, London B, Nerbonne JM. Functional consequences of elimination of i(to,f) and i(to,s): early afterdepolarizations, atrioventricular block, and ventricular arrhythmias in mice lacking Kv1.4 and expressing a dominant-negative Kv4 alpha subunit. Circ Res. 2000; 87: 73–79.[Abstract/Free Full Text]
8. An WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature. 2000; 403: 553–556.[CrossRef][Medline] [Order article via Infotrieve]
9. Patel SP, Parai R, Parai R, Campbell DL. Regulation of Kv4.3 voltage-dependent gating kinetics by KChIP2 isoforms. J Physiol. 2004; 557: 19–41.[Abstract/Free Full Text]
10. Patel SP, Campbell DL, Strauss HC. Elucidating KChIP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChIP2 isoform. J Physiol. 2002; 545: 5–11.[Abstract/Free Full Text]
11. Rosati B, Grau F, Rodriguez S, Li H, Nerbonne JM, McKinnon D. Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle. J Physiol. 2003; 548: 815–822.[Abstract/Free Full Text]
12. Zicha S, Xiao L, Stafford S, Cha TJ, Han W, Varro A, Nattel S. Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts. J Physiol. 2004; 561: 735–748.[Abstract/Free Full Text]
13. Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W, Lebel M, Cheng CW, Park CY, Pierce SA, Guerchicoff A, Pollevick GD, Chan TY, Kabir MG, Cheng SH, Husain M, Antzelevitch C, Srivastava D, Gross GJ, Hui CC, Backx PH, Bruneau BG. The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell. 2005; 123: 347–358.[CrossRef][Medline] [Order article via Infotrieve]
14. Rossow CF, Minami E, Chase EG, Murry CE, Santana LF. NFATc3-induced reductions in voltage-gated K⁺ currents after myocardial infarction. Circ Res. 2004; 94: 1340–1350.[Abstract/Free Full Text]
15. Tavi P, Pikkarainen S, Ronkainen J, Niemela P, Ilves M, Weckstrom M, Vuolteenaho O, Bruton J, Westerblad H, Ruskoaho H. Pacing-induced calcineurin activation controls cardiac Ca²⁺ signalling and gene expression. J Physiol. 2004; 554: 309–320.[Abstract/Free Full Text]
16. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential activation of transcription factors induced by Ca²⁺ response amplitude and duration. Nature. 1997; 386: 855–858.[CrossRef][Medline] [Order article via Infotrieve]
17. Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell. 1998; 93: 215–228.[CrossRef][Medline] [Order article via Infotrieve]
18. Bueno OF, Wilkins BJ, Tymitz KM, Glascock BJ, Kimball TF, Lorenz JN, Molkentin JD. Impaired cardiac hypertrophic response in calcineurin Abeta-deficient mice. Proc Natl Acad Sci U S A. 2002; 99: 4586–4591.[Abstract/Free Full Text]
19. Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, Jones F, Kimball TR, Molkentin JD. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res. 2004; 94: 110–118.[Abstract/Free Full Text]
20. Wilkins BJ, De Windt LJ, Bueno OF, Braz JC, Glascock BJ, Kimball TF, Molkentin JD. Targeted disruption of NFATc3, but not NFATc4, reveals an intrinsic defect in calcineurin-mediated cardiac hypertrophic growth. Mol Cell Biol. 2002; 22: 7603–7613.[Abstract/Free Full Text]
21. Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science. 1997; 275: 1930–1934.[Abstract/Free Full Text]
22. Ufret-Vincenty CA, Baro DJ, Santana LF. Differential contribution of sialic acid to the function of repolarizing K⁺ currents in ventricular myocytes. Am J Physiol Cell Physiol. 2001; 281: C464–C474.[Abstract/Free Full Text]
23. Xu H, Barry DM, Li H, Brunet S, Guo W, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res. 1999; 85: 623–633.[Abstract/Free Full Text]
24. London B, Guo W, Pan X, Lee JS, Shusterman V, Rocco CJ, Logothetis DA, Nerbonne JM, Hill JA. Targeted replacement of KV1.5 in the mouse leads to loss of the 4-aminopyridine-sensitive component of I(K,slow) and resistance to drug-induced QT prolongation. Circ Res. 2001; 88: 940–946.[Abstract/Free Full Text]
25. Dilly KW, Rossow CF, Votaw VS, Meabon JS, Cabarrus JL, Santana LF. Mechanisms underling variations in excitation-contraction coupling across the mouse left ventricular free wall. J Physiol. 2006; 572: 227–241.[Abstract/Free Full Text]
26. Perrier E, Perrier R, Richard S, Benitah JP. Ca²⁺ controls functional expression of the cardiac K⁺ transient outward current via the calcineurin pathway. J Biol Chem. 2004; 279: 40634–40639.[Abstract/Free Full Text]
27. Laurita KR, Katra R, Wible B, Wan X, Koo MH. Transmural heterogeneity of calcium handling in canine. Circ Res. 2003; 92: 668–675.[Abstract/Free Full Text]
28. Jia Y, Takimoto K. Mitogen-activated protein kinases control cardiac KChIP2 gene expression. Circ Res. 2006; 98: 386–393.[Abstract/Free Full Text]

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