Published online before print April 15, 2004, doi: 10.1161/01.RES.0000128406.08418.34
© 2004 American Heart Association, Inc.
Cellular Biology |
NFATc3-Induced Reductions in Voltage-Gated K+ Currents After Myocardial Infarction
From the Departments of Physiology and Biophysics (C.F.R., E.G.C., L.F.S.), Pathology (E.M., C.E.M.), and Medicine (E.M.), University of Washington, Seattle.
Correspondence to L.F. Santana, Department of Physiology and Biophysics, University of Washington, Box 357290, Seattle, WA 98195. E-mail santana{at}u.washington.edu
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Abstract |
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Reductions in voltage-activated K+ (Kv) currents may underlie arrhythmias after myocardial infarction (MI). We investigated the role of ß-adrenergic signaling and the calcineurin/NFAT pathway in mediating the reductions in Kv currents observed after MI in mouse ventricular myocytes. Kv currents were produced by the summation of 3 distinct currents: Ito, IKslow1, and IKslow2. At 48 hours after MI, we found a 4-fold increase in NFAT activity, which coincided with a decrease in the amplitudes of Ito, IKslow1, and IKslow2. Consistent with this, mRNA and protein levels of Kv1.5, 2.1, 4.2, and 4.3, which underlie IKslow1, IKslow2, and Ito, were decreased after MI. Administration of the ß-blocker metoprolol prevented the activation of NFAT and the reductions in Ito, IKslow1, and IKslow2 after MI. Cyclosporine, an inhibitor of calcineurin, also prevented the reductions in these currents after MI. Importantly, Kv currents did not change after MI in ventricular myocytes from NFATc3 knockout mice. Conversely, chronic ß-adrenergic stimulation or expression of an activated NFATc3 decreased Kv currents to a similar extent as MI. Taken together, these data indicate that NFATc3 plays an essential role in the signaling pathway leading to reduced Ito, IKslow1, and IKslow2 after MI. We propose that increased ß-adrenergic signaling after MI activates calcineurin and NFATc3, which decreases Ito, IKslow1, and IKslow2 via a reduction in Kv1.5, Kv2.1, Kv4.2, and Kv4.3 expression.
Key Words: calcineurin • arrhythmias • heart failure
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Introduction |
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Arrhythmias are a major cause of death after myocardial infarction (MI). Mounting evidence suggests that modification of the ventricular action potential (AP) underlies the increased probability of developing lethal arrhythmias after MI.1–5 Changes in the AP, including reductions in the rate of depolarization, peak depolarization,5 and an increase in the AP duration3 have been reported in regions proximal to and removed from the infarct zone. These changes in the AP are produced by reductions in the magnitudes of Na+6 and Kv1,2 currents as well as changes in Ca2+ signaling proteins7 of ventricular myocytes. Some of these changes can occur rapidly; decreased Kv currents have been reported 3 days after MI.1,2 However, the signaling mechanisms underlying these profound electrical changes in the heart are unclear.
Recent studies have suggested a role for increased ß-adrenergic receptor (ßAR) signaling in the development of the hypertrophic phenotype post-MI, including changes in Kv currents.8–10 Accordingly, catecholamine levels in the heart, as well as protein kinase A (PKA) activity in ventricular myocytes, have been found to increase after MI.9,10 Augmentation of PKA activity can elicit profound changes in myocardial Ca2+ signaling, including increased Ca2+ currents and sarcoplasmic reticulum (SR) Ca2+ release.11 Furthermore, chronic activation of PKA causes cardiac hypertrophy and increases the probability of arrhythmogenesis and sudden death.12
The Ca2+-activated phosphatase calcineurin has been hypothesized to play a pivotal role in translating altered Ca2+ signaling into changes in the transcription of genes linked to the development of hypertrophy and heart failure.13 On activation, calcineurin dephosphorylates NFAT, allowing translocation into the nucleus, whereupon it modulates the transcription of multiple genes. Expression of an activated form of NFATc4 is sufficient to induce robust hypertrophy in transgenic mice.13 However, NFATc3-null, but not NFATc4-null, mice exhibit decreased cardiac hypertrophy in response to calcineurin activation.14 These findings suggest that while NFATc4 can induce hypertrophy, it is NFATc3 that plays an essential role in the pathway leading to cardiac hypertrophy after calcineurin activation. A recent study found that calcineurin15 and NFATc316 are activated after MI and that inhibition of calcineurin prevented activation of this transcription factor and decreased cardiac hypertrophy post-MI. At present, however, it is unclear if calcineurin/NFATc3 signaling is involved in the electrical remodeling of the heart after MI.
In this study, we examined the role of ßAR stimulation, calcineurin, and NFATc3 in mediating reductions in Kv currents after MI. Our data suggest that increased ß-adrenergic signaling activates NFAT after MI. We propose that activation of NFATc3 decreases Kv currents via changes in gene expression. These findings suggest that ßARs, calcineurin, and NFATc3 play a crucial role in the pathway leading to reduced Kv currents post-MI.
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Methods |
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Generation of Myocardial Infarcts
Infarcts were generated as previously described17 in accordance to the guidelines of the Institutional Animal Care and Use Committee. NFATc3 knockout and NFAT reporter mice were kindly provided by Drs Laurie Glimcher (Harvard University, Cambridge, Mass) and Mercedes Rincón, (University of Vermont, Burlington), respectively.
Isolation, Short-Term Culture, and Adenoviral Infection of Adult Mouse Ventricular Myocytes
Ventricular myocytes from adult mice were isolated from regions remote to the MI (ie, right ventricle, noninfarcted section of the left ventricle and septum) 48 hours post-MI. For control experiments, cells were dissociated from regions of the heart similar to those used from infarcted hearts. Adenoviral infection and short-term culture of adult ventricular myocytes were performed as described elsewhere.18
Electrophysiology and Confocal Microscopy
Intracellular Ca2+ ([Ca2+]i) imaging was performed with a confocal microscope. Currents and membrane potentials were recorded using an Axopatch 200B amplifier.
Administration of CsA, FK506, and Metoprolol
Metoprolol (
350 mg/kg per day) was administered via drinking water.19 CsA (25 mg/kg per day) and FK506 (3 mg/kg twice per day) were administered intraperitoneally.
Real-Time Reverse-Transcription Polymerase Chain Reaction
Reverse-transcription (RT) was performed using the Superscript First-strand Synthesis system (Invitrogen). Real-time polymerase chain reaction (PCR) was performed with the TaqMan 5' nuclease assay and TaqMan Onestep PCR Mastermix.
Western Blots
Protein (50 µg) was loaded on a 4% to 15% Tris-HCl polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Blots were incubated with primary antibodies specific to Kv1.5, 2.1, 4.2, or 4.3, and then with horseradish peroxidase-conjugated secondary antibodies. Kv channel protein was quantified by densitometry.
Luciferase Assay
Luciferase activity was quantified using a commercially available kit (Promega).
Statistics
Data are presented as mean±SEM. Group comparisons were made using Student t test or ANOVA, which, if necessary, were followed-up by a Tukey test.
An expanded Methods section can be found in the online data supplement available at http://circres.ahajournals.org.
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Results |
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Changes in AP Duration and Whole-Cell [Ca2+]i Transients 48 Hours After MI
We examined [Ca2+]i and the AP of ventricular myocytes from control and infarcted hearts. For these experiments, ventricular myocytes from regions remote to the MI were isolated 48 hours after permanent ligation of the left anterior descending coronary artery (typically midway from the apex to base). Although we could not measure the infarct size in the same hearts from which myocytes were isolated, parallel experiments yielded infarcts that reproducibly averaged 38%±5% of the left ventricle.20 Figure 1A shows representative steady-state (1 Hz) AP and [Ca2+]i transient records obtained from control and MI myocytes. The peak and maximal rate of depolarization of the AP were similar in MI (peak=46.1±2.7 mV, dV/dtmax=253.8±25.1 V/s, n=16) and control cells (peak=48.6±1.6 mV, dV/dtmax=246.3±25.1 V/s, n=54; P>0.05). However, AP duration at 90% repolarization (APD90) was nearly 80% longer in MI (198.4±19.8 ms) than in control myocytes (109.5±5.6 ms, P<0.05; Figure 1B). We also found that the amplitude of the AP-evoked whole-cell [Ca2+]i transient was
1.7-fold higher in MI (935.1±55.7 nmol/L, n=18) than in control (548.3±45.4 nmol/L, n=25, P<0.05) myocytes.
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Reductions in Ito, IKslow1, and IKslow2 After MI
One mechanism by which APD could increase after MI is by a decrease in the Kv currents responsible for the repolarization of ventricular cells. Thus, we examined Kv currents in MI (n=26) and control (n=28) ventricular myocytes (Figure 2). Kv currents were evoked by 1.2-second depolarizing pulses from –90 mV to potentials ranging from –60 to +40 mV. The Kv currents evoked by this protocol activated rapidly and then decayed with time (Figure 2A). The decaying phase of the Kv currents recorded during depolarization to +40 mV could be fit with the sum of 2 exponential functions.21 At +40 mV, Kv currents from control cells had a fast (
fast) and slow (slow) time constant of inactivation of 57.2±9.8 ms and 531.9±33.8 ms, respectively. MI cells depolarized to the same potential produced Kv currents with similar fast (46.2±3.4 ms) and slow (616.6±58.3 ms) (P>0.05). However, we found that the amplitudes of the transient (Itran; defined as the difference between the peak and sustained current measured at the end of the pulse) and sustained (Isust; defined as the sustained current measured at the end of the pulse) components of Kv (IK) currents were smaller in MI than in control cells at most potentials examined (Figure 2A through 2C). At +40 mV, Itran was 70% smaller in MI (13.76±2.17 pA/pF) than in control (47.80±2.30 pA/pF) cells (P<0.05). At the same potential, Isust decreased by 33% after MI (control=16.88±1.11 versus MI=11.34±0.92 pA/pF, P<0.05). Cell capacitance, an indicator of cell surface area, increased by 20% 48 hours after MI (control=108.2±4.8 pF versus MI=130.2±8.7 pF, P<0.05).
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It is important to note that we found no significant differences in the voltage dependencies of conductance and steady-state inactivation of Itran and Isust in control and MI cells (P>0.05; data not shown). The voltage at which 50% of Itran and Isust were inactivated was similar in MI (Itran=–36.7±1.4 mV; Isust=–52.5±2.5 mV, n=12) and control cells (Itran=–38.2±2.4 mV; Isust=–49.6±3.5 mV, n=16) (P>0.05). Furthermore, Itran and Isust reached 50% of their maximal conductance at a similar voltage in MI (Ito=9.7±4.2 mV; Isust=–34.0±3.6 mV) and control (Ito=4.5±1.8 mV; Isust=–34.9±3.7 mV) cells (P>0.05). Taken together, these data suggest that a reduction in the amplitudes of Itran and Isust contribute to AP prolongation in mouse ventricular myocytes post-MI. The reductions in Itran and Isust were not results of changes in the voltage-dependencies of activation or inactivation of these currents post-MI.
The next series of experiments investigated the effects of MI on the currents that underlie Itran and Isust in mouse ventricular myocytes (Figure 2A). Three kinetically and pharmacologically distinct currents are thought to contribute to Itran and Isust.22,23 Itran is produced by the rapidly inactivating transient outward current (Ito), whereas a noninactivating current (IKslow2) contributes to Isust. A slowly inactivating current (IKslow1) contributes to Itran and Isust. Pharmacological tools were used to separate these currents. To isolate IKslow1, Kv currents were measured before and after the application of 50 µmol/L 4-aminopyridine (4-AP), which blocks this current, but has no effect on Ito or IKslow2.22 Subtraction of the currents recorded in the presence of 50 µmol/L 4-AP from the predrug traces results in the 4-AP-sensitive current or IKslow1. To obtain IKslow2, currents recorded during superfusion of a solution containing 50 µmol/L 4-AP and 25 mmol/L tetraethylammonium chloride (TEA), which blocks IKslow2, but has little effect on Ito,22 were subtracted from currents recorded in the presence of 4-AP. The TEA-sensitive current represents IKslow2. The remaining current recorded in the presence of 4-AP and TEA corresponds to Ito (defined here as the difference between the peak and sustained current measured at the end of the 1.2-second pulse). Itran, Isust, Ito, IKslow1, and IKslow2 were recorded from the same cells.
In agreement with the data shown in Figure 2B and 2C, Ito, IKslow1, and IKslow2 were significantly smaller after MI than in control cells (Figure 2D and 2F). At +40 mV, the amplitudes of Ito and IKslow1 were nearly 69% and 65% smaller after MI than in control cells, respectively. IKslow2 decreased by 30% 48 hours post-MI. These data suggest that the decrease in Itran observed in MI resulted from equivalent reductions in the magnitude of Ito and IKslow1. The decrease in Isust was produced by reductions in IKslow1 and IKslow2.
We examined the molecular mechanisms underlying the reductions in Ito, IKslow1, and IKslow2 observed post-MI. In the mouse ventricle, Ito is produced by a heterotetramer formed by Kv4.2 and Kv4.3 subunits.24 Kv1.5 and Kv2.1, in turn, underlie IKslow125 and IKslow2.26 Thus, we used real-time PCR to examine the mRNA levels of Kv1.5, Kv2.1, Kv4.2, and Kv4.3 in control and MI hearts (Figure 2G). Our data indicate that mRNA levels for Kv1.5, Kv2.1, Kv4.2, and Kv4.3 were 47%, 30%, 62%, and 41% lower (P<0.05) in MI (n=9 hearts) than in control (n=9 hearts) ventricles, respectively. Additional experiments using actinomycin D (see online data supplement) demonstrated that this decrease in mRNA did not result from an increase in the degradation rate of Kv1.5, Kv2.1, Kv4.2, or Kv4.3 transcripts in ventricular myocytes after MI. Consistent with our electrophysiological and mRNA data, Western blot analysis of fractionated ventricular membrane proteins (50 µg total protein) revealed that Kv1.5, Kv2.1, Kv4.2, and Kv4.3 protein decreased by 45%±10%, 48%±10%, 61%±13%, and 55%±5%, respectively, after MI (n=9 hearts, P<0.05) (Figure 2H). These data suggest that the decreases in Ito, IKslow1, and IKslow2 observed post-MI result from decreases in the expression of Kv1.5, Kv2.1, Kv4.2, and Kv4.3 mRNA and protein.
Administration of the ß-Blocker Metoprolol Prevents Reductions in Ito and Isust After MI
Administration of ß-blockers reduces the probability of lethal arrhythmias after MI.27 Because reductions in Kv currents have been linked to arrhythmogenesis, we investigated whether an increase in ßAR stimulation leads to the reduction in Ito, IKslow1, and IKslow2 observed post-MI. A testable prediction of this hypothesis is that ßAR blockers should minimize, or even prevent, the changes in Ito, IKslow1, and IKslow2 observed post-MI. Thus, the effects of the ßAR blocker, metoprolol, on Kv currents after MI were investigated (Figure 3).
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For these experiments, mice were given metoprolol beginning 24 hours before infarction (BB-MI). Control (noninfarcted; BB) animals received metoprolol for the same period of time. Metoprolol treatment prevented the decrease in Itran and Isust amplitudes (P>0.05) observed post-MI (Figure 3A and 3B). At +40 mV, the amplitudes of Itran in BB and BB-MI cells were 49.37±3.34 pA/pF (n=23) and 47.78±2.3 pA/pF (n=25), respectively. Accordingly, Ito, IKslow1, and IKslow2 were similar in BB-MI and control cells (Figure 3C through 3E). Consistent with this, we found, using real-time PCR and Western blot analysis, that metoprolol prevented the reductions in Kv1.5, Kv2.1, Kv4.2, and Kv4.3 mRNA (n=21 hearts, P>0.05) and protein (n=6 hearts, P>0.05) observed in the ventricles post-MI (Figure 3F and 3G).
Activation of NFAT After MI
The [Ca2+]i, mRNA, and protein data discussed above raise the intriguing possibility that increased Ca2+ signaling could underlie the changes in Kv channel expression observed post-MI via the activation of calcineurin/NFAT signaling. Indeed, analysis of the Kv1.5, Kv2.1, Kv4.2, and Kv4.3 genes revealed multiple putative NFAT binding elements (GGAAA) in the 5' untranslated region of these genes (see online data supplement). Thus, we tested the hypothesis that increased ßAR stimulation activates NFAT post-MI and thereby reduces the expression of Kv1.5, Kv2.1, Kv4.2, and Kv4.3. These experiments involved a transgenic NFAT reporter mouse (NFAT-luc), in which luciferase expression is controlled by multiple NFAT binding sites.28 We found that 48 hours after MI, luciferase activity in ventricular myocytes from areas remote to the MI was nearly 4-fold higher than in cells from the same region of noninfarcted hearts (NFAT-luc littermates) (control of 2.7±0.2 versus MI of 10.9±2.3 AU; n >100 cells from each of 3 hearts; P<0.05; Figure 4A). Interestingly, this increase in NFAT activity after MI was prevented by metoprolol treatment (BB-MI=2.3±0.3 AU; n >100 cells from each of 3 hearts; P>0.05). Metoprolol did not alter NFAT activity in noninfarcted myocytes (BB=2.7±0.2 AU; n >100 cells from 3 hearts; P<0.05). These data suggest that activation of ßARs post-MI leads to the activation of NFAT in mouse ventricular myocytes.
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CsA Prevents Ito and Isust Reductions After MI
NFAT is activated by the Ca2+-sensitive phosphatase calcineurin.13 Thus, we investigated whether activation of calcineurin was necessary to mediate the changes in Ito, IKslow1, and IKslow2 observed post-MI. To do this, Kv currents were recorded from cells dissociated from control (noninfarcted) and infarcted hearts from animals that had been administered a dose of CsA (25 mg/kg per day) shown, in vivo, to inhibit calcineurin activity in the heart.15 CsA treatment began 48 hours before infarction and continued until hearts were harvested. Noninfarcted mice were treated with a similar regimen. Figure 4B through 4F shows the current-voltage relationships of Kv currents from control (noninfarcted, CsA-treated mice; n=21), untreated MI (n=26), and cells dissociated from infarcted hearts treated with CsA (MI-CsA; n=18). CsA prevented the reduction of the amplitudes of Itran and Isust (P>0.05; Figure 4B and 4C) after MI. Like metoprolol, CsA also prevented the decreases in Ito, IKslow1, and IKslow2 observed post-MI (Figure 4D through 4F). FK506, another calcineurin inhibitor, also prevented the decrease in Kv currents after MI (n=7, P<0.05; data not shown). These data suggest that calcineurin plays a central role in mediating the changes in Ito, IKslow1, and IKslow2 observed post-MI.
NFATc3 Mediates the Reductions in Ito, IKslow1, and IKslow2 Observed After MI
To further examine the role of NFATc3 in mediating MI-induced reductions in Ito, Ikslow1, and Ikslow2, we performed experiments similar to those described above in NFATc3 knockout (NFATc3-KO) mice. Figure 5A shows representative Kv currents from ventricular myocytes dissociated from NFATc3–KO, infarcted NFATc3–KO (NFATc3–KO-MI), and infarcted wild-type (WT-MI) hearts. If activation of NFATc3 is necessary for the reduction in Ito, IKslow1, and IKslow2 observed post-MI, then there should be no reduction in these currents after MI in ventricular myocytes from NFATc3-KO mice. Supporting this hypothesis, Figure 5B through 5F show that although MI decreased Itran, Isust, IKslow1, IKslow2, and Ito in WT cells (WT-MI; n=26), the amplitudes of these currents were similar (P>0.05) in NFATc3–KO (n=26) and NFATc3–KO-MI cells (n=14). Consistent with these data, Kv1.5, Kv2.1, Kv4.2, and Kv4.3 mRNA (n=20 hearts) and protein (n=6 hearts) did not decrease (P>0.05) after MI in NFATc3–KO ventricles (Figure 5G and 5H). These data suggest that NFATc3 plays an obligatory role in the reductions of Ito, IKslow1, and IKslow2 observed 48 hours post-MI. Interestingly, a comparison of Kv currents from WT and NFATc3–KO cells revealed that Ito, but not IKslow1 or IKslow2, was increased in NFATc3–KO cells, thus suggesting a degree of basal regulation of Ito by NFATc3 (see online data supplement).
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Chronic ßAR Stimulation and a Constitutively Active NFATc3 Mimic the Effects of MI on Itran and Isust
To test if activation of NFATc3 was sufficient to account for the changes in Itran and Isust observed post-MI, we infected ventricular myocytes from uninfarcted WT mice with an adenoviral vector expressing a constitutively active EGFP-tagged NFATc3 mutant (
NFATc3; Figure 6). Control cells were infected with an adenoviral vector expressing EGFP only. As expected, cells expressing activated NFATc3 showed high levels of fluorescence in their nuclei (Figure 6A). The rate of inactivation of the composite Kv currents at +40 mV was similar in control (fast=43.1±8.4 ms, slow=623.8±107.9 ms, n=8) and NFATc3 cells (fast=44.7±5.8 ms, slow=519.5±67.5 ms; n=8, P>0.05). However, expression of NFATc3 reduced (P<0.05) the amplitudes of Itran and Isust at most voltages examined. Note that cells cultured for 48 hours in the presence of the ß-adrenergic agonist isoproterenol (ISO; 10 nmol/L) had Itran and Isust that were smaller than those from cells cultured for the same amount of time, but in the absence of ISO. Interestingly, ISO reduced Itran and Isust to the same extent as NFATc3 (n=6; P>0.05). These findings suggest that activation of NFATc3 and of ßARs reduce the amplitude of Kv currents in mouse ventricular myocytes.
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Discussion |
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In this study, we provide the first direct demonstration that activation of NFATc3 is essential in the pathway leading to decreased Kv currents post-MI. Our data suggest that increased ß-adrenergic signaling plays a pivotal role in the activation of NFATc3 after MI. We propose that activation of NFATc3 after MI downregulates Kv1.5, Kv2.1, Kv4.2, and Kv4.3 gene expression, thereby decreasing the amplitudes of Ito, IKslow1, and IKslow2 in mouse ventricular myocytes. The implications of these findings are discussed below.
AP and [Ca2+]i After MI
We found that decreased Ito, IKslow1, and IKslow2 accompanied AP prolongation post-MI. This is consistent with recent studies suggesting that Ito, IKslow1, and IKslow2 are responsible for the repolarization of the mouse ventricle.22,23 AP prolongation increases Ca2+ influx via L-type Ca2+ channels, SR Ca2+ load, and SR Ca2+ release during excitation-contraction coupling.29 A similar mechanism may underlie the larger [Ca2+]i transient observed here 48 hours post-MI. However, it is intriguing to speculate that increased ßAR signaling9 could also contribute to enhanced [Ca2+]i early after MI.
Changes in the maximal rate of depolarization and peak of the AP of control and MI cells were not observed, indicating that the Na+ current (INa) was not changed 48 hours post-MI.5 In support of this, we found that INa was similar in control and MI cells (Santana, Rossow, Minami, and Murry, unpublished data). Note, however, that changes in INa have been observed 5 days post-MI.6 Thus, decreased Kv currents appear to represent an earlier event in the progression of AP changes post-MI.
NFATc3 and Kv Expression in Ventricular Myocytes
The data suggesting that NFATc3 is an essential component of the signaling pathway mediating the changes in Ito, IKslow1, and IKslow2 after MI are compelling. First, we found that NFAT activity increased nearly 4-fold post-MI. This finding is consistent with a recent study showing NFATc3 hypophosphorylation 4 weeks post-MI.16 Second, prevention of NFATc3 activation by pharmacological (metoprolol and presumably by CsA and FK506) or genetic (NFATc3–KO mice) interventions prevented the decreases in Ito, IKslow1, and IKslow2 observed after MI. Third, expression of an activated form of NFATc3 mimics the actions of MI and chronic ßAR stimulation on Kv currents in mouse ventricular myocytes.
Our functional, mRNA and protein data suggest that reduced expression of Kv1.5, Kv2.1, Kv4.2, and Kv4.3 transcripts and proteins is the likely mechanism underlying the decreases in Ito, IKslow1, and IKslow2 post-MI. We found multiple-consensus NFAT-binding elements within the 5' untranslated region of the Kv1.5, Kv2.1, Kv4.2, and Kv4.3 genes. Thus, binding of NFATc3 to these genes could possibly alter their expression. This view is supported by a recent study showing that NFATc3 can downregulate the transcription of CD154, CDK4, IL-4, and IL-5 genes.30 NFATc3 can therefore have a dual role as inducer or repressor of gene transcription.
An intriguing finding in this study is that activation of NFATc3 after MI downregulated Ito and IKslow1 to a larger extent than IKslow2. The molecular mechanisms underlying this difference are unclear, however. We did not detect a significant difference in the relative decrease of Kv4.3, Kv4.2, Kv2.1, and Kv1.5 mRNA and protein post-MI. The difference in the magnitude of the decrease in currents and Kv proteins post-MI could reflect changes in the expression of accessory subunits (eg, KChIPs, KChAPs, Kvßs), trafficking and/or posttranslational processing of these channels. This is important because there are pronounced differences in the amplitude and kinetics of Ito within different regions of the mouse heart.23,31 Indeed, the Itos reported here have a relatively large amplitude and are fully inactivated by 200 ms, which suggest that they likely represent the fast Ito described in cells from the ventricular epicardium31 and in some cells from the septum.23 IKslow1 is also differentially expressed within the mouse ventricle. Interestingly, Xu et al23 found that IKslow1 was larger in cells with larger fast Ito. Future experiments should examine a possible role for NFATc3 in maintaining the regional heterogeneity of Ito and IKslow1 in the heart.
Ca2+ Signaling and NFATc3 Activity in the Heart
In a recent study by Sah et al,32 overexpression of a truncated form of Kv4.2 decreased Ito and prolonged the AP of ventricular myocytes of transgenic mice. They found that AP prolongation was associated with increased [Ca2+]i and also with activation of calcineurin. These experiments suggested that calcineurin is downstream of changes in Kv currents. However, we overexpressed a constitutively active NFATc3 and found decreased Kv currents, indicating Kv channel transcription is downstream of NFATc3 signaling (Figure 6). Although these 2 studies may initially seem at odds, we suggest a signaling model that is consistent with both observations (Figure 7). We propose that early after MI, ßAR-induced changes in [Ca2+]i result in calcineurin/NFATc3 activation which, in turn, results in decreased Kv channel expression. Reduced Kv channel expression then leads to decreased Kv currents and hence to AP prolongation, which increases [Ca2+]i. In our model, ßAR, calcineurin, and NFATc3 form part of a positive feedback loop that could be activated by increasing [Ca2+]i, directly activating NFATc3, and so on. Initiation of this feedback loop could be prevented by the administration of ß-blockers or calcineurin inhibitors.
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Comparison With Other Studies
Our hypothesis that ßARs and NFATc3 play a central role in the downregulation of Ito, IKslow1, and IKslow2 post-MI is supported by recent work. Zhang et al33 suggested that chronic activation of ßARs reduce the amplitude of delayed rectifier K+ currents in cultured guinea pig ventricular myocytes. Furthermore, it was reported that mice overexpressing calcineurin show early downregulation of Kv2.1 and Kv1.5 protein levels.34 Hearts overexpressing calcineurin also had lower Kv4.2 protein expression than controls, but only after the development of heart failure.34 A recent study by Deng et al15 used calcineurin inhibition by CsA as a tool to evaluate the role of this phosphatase in mediating the changes in Kv currents observed in the rat post-MI. Consistent with our results and those of Petrashevskaya et al,34 they found that CsA administration inhibited calcineurin and prevented the reductions in Kv4.2 and Kv2.1 expression observed post-MI. These findings support the hypothesis that activation of NFATc3 by calcineurin could underlie electrical remodeling of the heart after injury.
In addition to inducing Kv channel downregulation, MI causes hypertrophy. Two recent studies suggested that prevention of calcineurin activation using CsA15 or by overexpressing the inhibitory calcineurin-interacting protein-116 decreased ventricular hypertrophy by up to 40% after MI. By comparison, our data indicate that preventing NFATc3 activation by pharmacological or genetic means completely prevents Kv current remodeling post-MI. Given that calcineurin/NFAT inhibition only partially blocks hypertrophy, whereas Kv downregulation is completely abrogated, we speculate that there may be less redundancy in the pathways leading to Kv downregulation.
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Summary |
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Our results indicate that ßARs, calcineurin, and NFATc3 are part of a signaling pathway that reduces Kv currents post-MI. We propose that calcineurin reduces these currents by decreasing the expression of Kv1.5, Kv2.1, Kv4.2, and Kv4.3 channels via the activation of NFATc3. These findings provide a mechanistic model for the therapeutic use of ß-blockers to reduce arrhythmogenic changes in Kv currents after MI.
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Acknowledgments |
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This work was supported by National Institutes of Health (HL70556, HL061553, NRSA802378, and HL003712) and American Heart Association (0320026Z) grants. We thank Drs Greg Amberg, Marv E. Adams, and Carmen Ufret for reading this manuscript. We thank Erin Sylvester for her technical assistance.
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Footnotes |
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Received August 12, 2003; revision received April 5, 2004; accepted April 7, 2004.
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References |
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