Differential Calcineurin/NFATc3 Activity Contributes to the Ito Transmural Gradient in the Mouse Heart
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Charles F. Rossow, Keith W. Dilly, Luis
参见附件。
the Department of Physiology and Biophysics, University of Washington, Seattle.
Abstract
Kv4 channels are differentially expressed across the mouse left ventricular free wall. Accordingly, the transient outward K+ current (Ito), 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 Ito 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 Ito 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 Ito 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 Ito function, in the mouse left ventricle.
Key Words: signal transduction arrhythmias voltage-gated potassium currents calcium gene regulation
Introduction
The transient outward K+ current (Ito) 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 Ito.1 In human and canine, which do not express Kv4.2, Ito is produced by Kv4.3 subunits. A distinctive feature of the mammalian heart is that Ito function varies throughout the myocardium.2,3 Indeed, there is a striking difference in the amplitude of Ito between left ventricular epicardial (EPI) than in endocardial (ENDO) cells,2,3 with Ito being larger in EPI than in ENDO myocytes.2,4,5 This transmural gradient of Ito function is important for normal ventricular repolarization.4,6,7
Previous work examining the mechanisms underlying regional differences in Ito 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 Ito.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 expression3,11,12 is thought to control regional differences in Ito 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 Ito 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 Ito function in ventricular myocytes.14 NFATc3 decreased Ito 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 Ito 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 Ito function in ENDO cells. Importantly, we show that NFATc3 expression was necessary for differential Ito 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 Ito function in the mouse myocardium.
Materials and Methods
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
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).
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).
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 subunit of calcineurin A—the predominant calcineurin isoform in heart18—in EPI and ENDO. As shown in Figure 2B, calcineurin A protein is expressed to a similar extent in EPI and ENDO (n=5 hearts, P>0.05). Thus, increased calcineurin A 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 -actin mRNA was also measured in these tissues. Consistent with our hypothesis, we found that luciferase, but not -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.
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 -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 (GSK3).21 On phosphorylation, GSK3 (p-GSK3) is activated, thus phosphorylating conserved serines within the NFAT N terminus that are required for its nuclear export. Thus, we examined GSK3 activity in ENDO and EPI (Figure 3C). We found that the levels of total GSK3 (ie, inactive kinase) and p-GSK3 (ie, active kinase) protein were similar in ENDO and in EPI cells (n=5, P>0.05), indicating that GSK3 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-GSK3, underlies higher NFAT activity in ENDO than in EPI.
NFATc3 Is Required for Establishing the Ito 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 Ito gradient. A testable prediction of this hypothesis is that mice lacking NFATc3 expression (NFATc3–/–) would, at a minimum, have a less pronounced gradient of Ito function across the ventricular wall.
Ito currents were recorded in EPI and ENDO cells isolated from wild-type (WT) and NFATc3–/– hearts (Figure 4A). Ito was isolated from other voltage-gated K+ currents in these cells by pharmacological means, as previously described.22 Ito 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.
To begin, we recorded Ito in WT ENDO and EPI myocytes. As previously reported,5 Ito was larger in WT EPI than in ENDO cells at most voltages examined (Figure 4A and 4B). Surprisingly, we found that Ito was similar in EPI and ENDO cells from NFATc3–/– hearts. Note that equalization of Ito amplitudes between ENDO and EPI cells from NFATc3–/– hearts was produced by increased Ito function in ENDO cells as opposed to decreased Ito 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 Ito across the left ventricular wall.
We investigated the molecular mechanisms by which NFATc3 regulates Ito function. In mouse ventricular myocytes, Ito 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 Ito 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). -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 -actin) was similar in WT and NFATc3–/– ENDO and EPI cells (P>0.05). These findings suggest that differential expression of Ito between EPI and ENDO results from NFATc3-dependent regional differences in Kv4.2 expression.
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 Ito 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 Ito function across the left ventricular wall.
EPI and ENDO Cells With Matching Calcineurin Activities Have Similar Ito Amplitudes
After having established that greater basal NFATc3 activity decreased Kv4.2 expression and Ito function in ENDO cells, we investigated the relationship between calcineurin activity and Ito in EPI and ENDO cells. If calcineurin negatively regulated Ito (via NFATc3), as we have proposed, then a sustained increase in calcineurin activity in EPI cells should reduce the amplitude of Ito in these cells. Conversely, Ito 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 Ito. 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 Ito in ENDO and EPI cells that had been cultured for 24 hours under these conditions (ie, no pacing). For comparison, Ito 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 Ito (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 Ito in EPI (acutely dissociated and cultured cells) and ENDO cells with matching [Ca2+]i was not different.
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 Ito 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, Ito was similar in cultured and acutely dissociated ENDO cells. These data indicate that increasing [Ca2+]i reduces Ito in EPI cells. Furthermore, our data indicate that the higher [Ca2+]i in ENDO cells in vivo contributes to the smaller Ito observed in these cells.
We investigated whether the observed reduction of Ito 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 Ito 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 Ito. Taken together, our data suggest that the amplitude of Ito depends on the level of diastolic [Ca2+]i and calcineurin activity in left ventricular myocytes. Accordingly, we propose that the transmural gradient of Ito 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 Ito 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.
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 IK,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 IK,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
We provide evidence that regional differences in calcineurin/NFATc3 signaling contribute to heterogeneous Ito 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 Ito 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 Ito in ENDO cells. We,14 and others,26 have demonstrated that increased calcineurin activity is transduced into a reduction of Ito through activation of NFATc3. The absence of a transmural Ito gradient in NFATc3–/– ventricles strongly supports this hypothesis and indicates the necessity of this transcription factor for regional variations in Ito. Careful analysis of our NFATc3–/– data shows that Ito homogeneity in these hearts resulted from increased Ito in ENDO cells; there were no differences in Ito 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 GSK3-dependent phosphorylation. Our data suggest that increased import, not decreased GSK3-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 IK,slow and Ito 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, IK,slow is similar in these cells.13 Our data suggesting that Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChIP2 expression and Ito function depend on the relative activity of calcineurin/NFAT signaling in ventricular myocytes strongly support this model.
The transmural Ito gradient in canine and human hearts is produced by differential Kv4.312 and/or KChIP23,11 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito function during pathological conditions, they do not appear to contribute to differential Ito 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 Ito 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.
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the Department of Physiology and Biophysics, University of Washington, Seattle.
Abstract
Kv4 channels are differentially expressed across the mouse left ventricular free wall. Accordingly, the transient outward K+ current (Ito), 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 Ito 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 Ito 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 Ito 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 Ito function, in the mouse left ventricle.
Key Words: signal transduction arrhythmias voltage-gated potassium currents calcium gene regulation
Introduction
The transient outward K+ current (Ito) 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 Ito.1 In human and canine, which do not express Kv4.2, Ito is produced by Kv4.3 subunits. A distinctive feature of the mammalian heart is that Ito function varies throughout the myocardium.2,3 Indeed, there is a striking difference in the amplitude of Ito between left ventricular epicardial (EPI) than in endocardial (ENDO) cells,2,3 with Ito being larger in EPI than in ENDO myocytes.2,4,5 This transmural gradient of Ito function is important for normal ventricular repolarization.4,6,7
Previous work examining the mechanisms underlying regional differences in Ito 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 Ito.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 expression3,11,12 is thought to control regional differences in Ito 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 Ito 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 Ito function in ventricular myocytes.14 NFATc3 decreased Ito 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 Ito 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 Ito function in ENDO cells. Importantly, we show that NFATc3 expression was necessary for differential Ito 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 Ito function in the mouse myocardium.
Materials and Methods
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
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).
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).
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 subunit of calcineurin A—the predominant calcineurin isoform in heart18—in EPI and ENDO. As shown in Figure 2B, calcineurin A protein is expressed to a similar extent in EPI and ENDO (n=5 hearts, P>0.05). Thus, increased calcineurin A 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 -actin mRNA was also measured in these tissues. Consistent with our hypothesis, we found that luciferase, but not -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.
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 -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 (GSK3).21 On phosphorylation, GSK3 (p-GSK3) is activated, thus phosphorylating conserved serines within the NFAT N terminus that are required for its nuclear export. Thus, we examined GSK3 activity in ENDO and EPI (Figure 3C). We found that the levels of total GSK3 (ie, inactive kinase) and p-GSK3 (ie, active kinase) protein were similar in ENDO and in EPI cells (n=5, P>0.05), indicating that GSK3 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-GSK3, underlies higher NFAT activity in ENDO than in EPI.
NFATc3 Is Required for Establishing the Ito 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 Ito gradient. A testable prediction of this hypothesis is that mice lacking NFATc3 expression (NFATc3–/–) would, at a minimum, have a less pronounced gradient of Ito function across the ventricular wall.
Ito currents were recorded in EPI and ENDO cells isolated from wild-type (WT) and NFATc3–/– hearts (Figure 4A). Ito was isolated from other voltage-gated K+ currents in these cells by pharmacological means, as previously described.22 Ito 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.
To begin, we recorded Ito in WT ENDO and EPI myocytes. As previously reported,5 Ito was larger in WT EPI than in ENDO cells at most voltages examined (Figure 4A and 4B). Surprisingly, we found that Ito was similar in EPI and ENDO cells from NFATc3–/– hearts. Note that equalization of Ito amplitudes between ENDO and EPI cells from NFATc3–/– hearts was produced by increased Ito function in ENDO cells as opposed to decreased Ito 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 Ito across the left ventricular wall.
We investigated the molecular mechanisms by which NFATc3 regulates Ito function. In mouse ventricular myocytes, Ito 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 Ito 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). -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 -actin) was similar in WT and NFATc3–/– ENDO and EPI cells (P>0.05). These findings suggest that differential expression of Ito between EPI and ENDO results from NFATc3-dependent regional differences in Kv4.2 expression.
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 Ito 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 Ito function across the left ventricular wall.
EPI and ENDO Cells With Matching Calcineurin Activities Have Similar Ito Amplitudes
After having established that greater basal NFATc3 activity decreased Kv4.2 expression and Ito function in ENDO cells, we investigated the relationship between calcineurin activity and Ito in EPI and ENDO cells. If calcineurin negatively regulated Ito (via NFATc3), as we have proposed, then a sustained increase in calcineurin activity in EPI cells should reduce the amplitude of Ito in these cells. Conversely, Ito 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 Ito. 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 Ito in ENDO and EPI cells that had been cultured for 24 hours under these conditions (ie, no pacing). For comparison, Ito 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 Ito (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 Ito in EPI (acutely dissociated and cultured cells) and ENDO cells with matching [Ca2+]i was not different.
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 Ito 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, Ito was similar in cultured and acutely dissociated ENDO cells. These data indicate that increasing [Ca2+]i reduces Ito in EPI cells. Furthermore, our data indicate that the higher [Ca2+]i in ENDO cells in vivo contributes to the smaller Ito observed in these cells.
We investigated whether the observed reduction of Ito 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 Ito 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 Ito. Taken together, our data suggest that the amplitude of Ito depends on the level of diastolic [Ca2+]i and calcineurin activity in left ventricular myocytes. Accordingly, we propose that the transmural gradient of Ito 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 Ito 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.
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 IK,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 IK,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
We provide evidence that regional differences in calcineurin/NFATc3 signaling contribute to heterogeneous Ito 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 Ito 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 Ito in ENDO cells. We,14 and others,26 have demonstrated that increased calcineurin activity is transduced into a reduction of Ito through activation of NFATc3. The absence of a transmural Ito gradient in NFATc3–/– ventricles strongly supports this hypothesis and indicates the necessity of this transcription factor for regional variations in Ito. Careful analysis of our NFATc3–/– data shows that Ito homogeneity in these hearts resulted from increased Ito in ENDO cells; there were no differences in Ito 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 GSK3-dependent phosphorylation. Our data suggest that increased import, not decreased GSK3-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 IK,slow and Ito 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, IK,slow is similar in these cells.13 Our data suggesting that Kv1.5, Kv2.1, Kv4.2, Kv4.3, and KChIP2 expression and Ito function depend on the relative activity of calcineurin/NFAT signaling in ventricular myocytes strongly support this model.
The transmural Ito gradient in canine and human hearts is produced by differential Kv4.312 and/or KChIP23,11 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito 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 Ito function during pathological conditions, they do not appear to contribute to differential Ito 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 Ito 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.
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