Mechanisms Underlying Rate-Dependent Remodeling of Transient Outward Potassium Current in Canine Ventricular Myocytes
Transient outward K+ current (Ito) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac Ito and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37°C) were paced continuously at 1 or 3 Hz for 24 hours. Ito was recorded with whole-cell patch clamp. The 3-Hz pacing reduced Ito by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by ≈30% and ≈40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced Ito downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented Ito downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca2+i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na+/Ca2+ exchange and sarcoplasmic reticulum Ca2+-ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented Ito reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates Ito expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent Ito control.
Sudden cardiac death caused by ventricular tachycardia or fibrillation is an important contributor to mortality in congestive heart failure (CHF) patients.1 Rapid heart-rhythms can impair cardiac function and patients with “tachycardiomyopathy” are at risk of sudden cardiac death.2 Chronic ventricular tachypacing in experimental animals produces a dilated cardiomyopathy that mimics clinical tachycardiomyopathy and is often used as an experimental model to study CHF-related cardiac remodeling.3 Changes in cardiac ion channel transport are important components of this remodeling, and extensive evidence suggests that these ion transport changes are crucial contributors to the pathogenesis of CHF-related ventricular tachyarrhythmias and sudden death.3,4 Among the most ubiquitous changes are alterations in the transient outward K+ current (Ito),3 which play potentially important roles in repolarization abnormalities,5,6 cardiac dysfunction,7 and arrhythmogenesis.6 Although CHF itself can cause Ito downregulation, the possibility that rapid cardiomyocyte rate per se can alter Ito function has not been examined. This possibility cannot be examined directly in vivo, because sustained tachycardia causes a CHF syndrome, with major attendant hemodynamic, neurohumoral, and autonomic nervous system alterations, making it impossible to discern the role of heart rate per se. In the present study, we used a model of paced adult canine ventricular cardiomyocytes to determine: (1) whether increases in firing rate alter Ito; and if so, (2) the signaling systems involved.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
In Vitro Cellular-Pacing Model
Animal care procedures followed NIH guidelines. Adult male mongrel dogs (20 to 37 kg, n=114) were anesthetized with pentobarbital (30 mg/kg IV). Epicardial cardiomyocytes were isolated as previously described.8 After isolation, cells were kept in medium 199 and centrifuged (500 rpm, 1 minute, 25°C). Cell pellets were resuspended and plated on laminin-coated glass coverslips (for electrophysiology or immunohistochemistry) or 4-well rectangular petri dishes (Western blot or real-time RT-PCR). After a 4-hour preincubation, cells were divided into groups for parallel study in each experimental protocol and were electrically paced with 5-ms pulses at 1 or 3 Hz (1- and 3-Hz paced cells, respectively) for 24 hours, unless otherwise indicated. After pacing, cells were kept in a high-K+ storage solution at 4°C for electrophysiological studies or were fast-frozen at −80°C for biochemical studies.
Ito was recorded at 36±0.5°C (for details see the online data supplement, expanded Materials and Methods section). Currents are expressed as current densities (normalized to cell capacitance). Resting potentials, compensated series resistances, cell capacitances, and cell dimensions were similar for 1- and 3-Hz groups (online data supplement, Table I). Correction for liquid junction potentials (which averaged ≈10 mV) was applied only for resting potential and reversal potential values.
Total RNA was extracted from 1- and 3-Hz paced cells with TRIzol. Real-time reverse transcription (RT)–quantitative PCR was performed with TaqMan assays for Kv4.3, Kv1.4, and KChIP2 with 18S ribosomal RNA as the internal control.
Membrane protein fractions were isolated from 1- and 3-Hz paced cells incubated without (CTL) or with KN93 or KN92. Proteins were separated on 8% or 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes. Blots were probed with primary antibodies against GAPDH (internal control for protein loading), Kv4.3, KChIP2, phospholamban (PLB) phosphorylated at threonine 17, total PLB, calcineurin, Na+/Ca2+ exchanger, ryanodine receptor, sarcoplasmic reticulum Ca2+-ATPase, nuclear factor of activated T cells (NFAT)c3, and NFATc4.
After 24-hour pacing, cells were washed with culture medium, then fixed with 2% paraformaldehyde and washed 3 times (5 minutes each) with PBS. After blocking and permeabilization (2% normal donkey serum and normal goat serum, along with 0.2% Triton X-100 for 1 hour), cells were incubated overnight at 4°C with primary antibodies for NFATc3 (1:200, mouse monoclonal) and NFATc4 (1:100, rabbit polyclonal) in PBS containing 1% normal donkey serum and 1% normal goat serum and 0.05% Triton, followed by 3 washes and secondary antibody (donkey anti-mouse Alexa-547 and goat anti-rabbit Alexa-488) incubation. Cells were then treated with RNaseA (100 μg/mL) and incubated with ToPro3 (1 μmol/L, for nuclear contour definition). Confocal microscopy was performed with a Zeiss LSM-510 system. Images were deconvolved using measured point spread functions. Nuclear and cytosolic NFATc3 and NFATc4 staining densities were determined as the sum of the pixels within each region normalized to region area. Measurements were repeated in 5 Z-stacks showing the maximum nuclear area in each cell.
Paced cell samples were collected after 6 hours of 1- or 3-Hz CTL, 3-Hz+KN93, or 3-Hz+KN92 (culture media containing 1 μmol/L KN93 or KN92) pacing based on preliminary studies showing peak activity after 6-hour 3-Hz pacing. Calcineurin activity was assessed with the Calcineurin Cellular Activity Assay Kit (Calbiochem).
Data Acquisition and Analysis
Clampfit 6.0 (Axon) and GraphPad Prism 3.0 were used for data analysis; curve fitting was performed with nonlinear least-square algorithms. Group comparisons were performed with paired or unpaired Student t tests or repeated-measures ANOVA with Bonferroni-corrected t tests or Dunnett’s tests. A 2-tailed P<0.05 indicated statistical significance; group data are expressed as means±SEM.
Rapid Rates Downregulate Ito and Kv4.3
Representative Ito recordings from cells paced at 1 Hz (to mimic normal resting heart rates) and 3 Hz (to mimic tachycardia) are shown in Figure 1A. Cells paced at 3 Hz showed smaller Ito with otherwise similar morphology versus 1-Hz paced cells and had significantly smaller Ito densities over a wide range of voltages, with an ≈45% reduction (Figure 1B). Ito inactivation kinetics were well fitted by biexponential relations, with inactivation time constants not different between 1- and 3-Hz cells (Figure 1C). The voltage dependence of Ito inactivation was studied with a 2-pulse protocol (Figure 1D, inset). Boltzmann relation fits showed no differences between 1- and 3-Hz: V1/2 values and slope factors averaged −39.9±2.8 and −3.8±0.1 mV, respectively, in 1-Hz (n=6) and −39.8±1.8 and −4.1±0.1 mV in 3-Hz cells (n=7, P=NS versus 1-Hz). Ito activation voltage dependence was assessed based on the relation Iv=Imax(V−Vr)(Gv/Gmax), where Iv and Gv are current and conductance at voltage V; Imax and Gmax are maximum current and conductance; and Vr is the reversal potential. Vr was determined by analyzing tail currents after 2.2-ms depolarizations to +50 mV and averaged −75.9±0.5 mV in 1-Hz (n=5) and −75.4±2.3 mV in 3-Hz cells (n=5; P=NS). Ito activation V1/2 averaged 8.9±0.6 mV in 1-Hz (n=9) and 9.7±1.5 mV in 3-Hz cells (n=8, P=NS). Ito reactivation (2-pulse protocol, Figure 1E) was well fitted by biexponential relations. Recovery time constants (τ) averaged 30±2 ms (τ fast) and 130±21 ms (τ slow) in 1-Hz (n=7) and 29±3 (τ fast) and 149±12 ms (τ slow) in 3-Hz cells (n=8, P=NS versus 1-Hz). Ito frequency dependence based on steady-state currents at 0.1, 0.5, 1, 2, and 5 Hz (100-ms pulses from −80 to +50 mV) was not different between 1- and 3-Hz cells (supplemental Figure I). To assess Ito downregulation in vivo, we tachypaced 4 dogs at 240 bpm for 24 hours and compared Ito on freshly isolated cardiomyocytes from tachypaced and CTL dogs. The results (supplemental Figure II) show significant decreases in Ito, consistent with in vitro observations.
To address the potential mechanisms underlying Ito downregulation, we first assessed mRNA and protein expression of potential underlying subunits: Kv4.3, Kv1.4, and KChIP2. The 3-Hz pacing significantly downregulated Kv4.3 mRNA (Figure 2A). Kv1.4 and KChIP2 mRNA expression was unaffected by 3-Hz pacing. Figure 2B shows examples of Kv4.3, KChIP2, and GAPDH immunoblots (top) and overall mean±SEM protein expression (bottom). Consistent with mRNA results, 3-Hz pacing downregulated Kv4.3 protein expression by ≈40%, whereas KChIP2 protein expression was unchanged.
These data indicate that rapid firing rates reduce Ito density through downregulation of Kv4.3 mRNA and protein. We next determined whether Ito downregulation requires cardiomyocyte mechanical activity and associated metabolic demands or whether electric activity is sufficient for downregulation. Blebbistatin (5 μmol/L), an excitation–contraction uncoupler with minimal direct electrophysiological actions,9 was added to the culture medium during 24-hour pacing at 1 and 3 Hz. Ca2+ transient activity in the absence of cell shortening confirmed cell capture during electromechanical uncoupling, as previously described.10 Supplemental Figure IIIA shows Ito recordings from cells studied in parallel with 1- and 3-Hz pacing, with and without blebbistatin, which failed to prevent rate-induced Ito downregulation (supplemental Figure IIIB). We then addressed the possibility that the results of 3-Hz stimulation could be attrib-utable to direct effects of larger total durations of electric be attributable to direct effects of larger total durations of electrical field stimulation. In parallel experiments, we subjected cells to 1- and 3-Hz pacing with 3-ms stimuli, as well as to 1-Hz stimulation with 9-ms stimuli (to provide the same total field stimulation duration as 3-Hz 3-ms stimuli), keeping stimulus intensities constant. As shown in supplemental Figure IV, 1-Hz stimulation with 9-ms pulses failed to reproduce the effects of 3-Hz 3-ms stimuli. We next determined whether downregulation of Ito requires angiotensin II receptor stimulation. Cells were subjected to 24 hours of 1- or 3-Hz pacing in the presence of the type-1 angiotensin II receptor antagonist valsartan (1 μmol/L). Valsartan failed to alter Ito rate regulation (supplemental Figure VA and VB). We then turned to investigate candidate Ca2+-dependent signal-transduction mechanisms for rate-dependent Ito regulation.
Role of Ca2+ Entry and Calmodulin
Intracellular [Ca2+] and Ca2+ binding to calmodulin are dynamic, changing on a beat-to-beat basis in cardiomyocytes.11 If [Ca2+]i changes are important in mediating the Ito frequency response, suppressing activation-related Ca2+ entry through ICaL should prevent rate-related Ito downregulation. Figure 3A and B show Ito recorded from cells cultured during 1- or 3-Hz pacing, in the presence of nimodipine (0.5 μmol/L, which decreased ICaL by ≈75%; supplemental Figure VI) or matching vehicle (CTL). The Ito-suppressant effect of 3-Hz pacing was eliminated by ICaL blockade (Figure 3C). We then incubated cells with W-7 (1 μmol/L, to inhibit calmodulin) or vehicle and repeated these studies with 2 other calmodulin antagonists, A-7 (at 1 and 5 μmol/L) and W-13 (40 μmol/L), along with its inactive analog W-12 (40 μmol/L), because of potential concerns about the efficacy and specificity of W-7. Calmodulin inhibition prevented Ito downregulation in 3-Hz cells (Figure 3D and supplemental Figure VII). Thus, Ca2+-dependent calmodulin function is implicated in rate-dependent Ito downregulation.
Role of CaMKII
High-frequency activation of Ca2+ transients increases CaMKII activity.12 To determine whether CaMKII activity is increased by 3-Hz pacing, we determined CaMKII-mediated Thr17 phosphorylation of PLB in cells cultured with vehicle (CTL), KN93 (1 μmol/L, a CaMKII inhibitor), or equivalent concentrations of the inactive analog KN92; preliminary experiments showed that 1 μmol/L KN93 had no effect on ICaL (supplemental Figure VIII). CaMKII-phosphorylated PLB expression was significantly increased in 3-Hz paced cells (Figure 4A and B; for expanded Western blots see, supplemental Figure IXA), although total PLB expression was unaltered. For cells paced at 1 or 3 Hz in the presence of KN93 to inhibit CaMKII activity, there were no differences in Thr17 PLB phosphorylation (Figure 4C and 4D). Cells paced at 3 Hz in the presence of KN92 showed increased CaMKII PLB phosphorylation similar to CTLs (Figure 4E and 4F).
To obtain functional evidence for CaMKII activation, we studied the potential effect of CaMKII hyperphosphorylation of PLB, which should enhance the rate of removal of cytosolic Ca2+ via sarcoplasmic reticulum Ca2+ uptake (by removing PLB inhibition of sarcoplasmic reticulum Ca2+-ATPase function). Ca2+ transients were recorded with Indo 1-acetoxymethyl ester as previously described.13 Ca2+ transients recorded at a 1000-ms cycle length from 1- and 3-Hz paced cells are shown in Figure 4G. The Ca2+ transient decay time constant was significantly decreased in 3-Hz paced cells (Figure 4H; time constants averaged 345±35 ms [n=6] in 3-Hz cells versus 454±30 ms in 1-Hz cells [n=8]; P=0.037). Differences in Ca2+ transient decay time could also be attributable to alterations in other Ca2+-handling proteins. The expression of other important Ca2+-handling proteins was assessed (supplemental Figure X) and showed no effect of 3-Hz pacing.
To determine whether increased CaMKII activity contributes to the Ito-suppressing effect of 3-Hz pacing, Ito was recorded from cells exposed to vehicle (CTL), KN93 (1 μmol/L), or KN92 (1 μmol/L) during 24-hour 1- or 3-Hz pacing. Figure 5A and B show original recordings of Ito on depolarization to +40 mV from 1- and 3-Hz paced cells. CaMKII inhibition by KN93 prevented 3-Hz pacing-induced Ito reduction but had no effect on Ito in 1-Hz paced cells (Figure 5C). KN92 had no protective effect on Ito downregulation. We then studied the effects of CaMKII inhibition on Kv4.3 and KChIP2 protein expression (Figure 5D; expanded Western blots in supplemental Figure IXB). Whereas, in the presence of CaMKII inhibition with KN93, Kv4.3 protein expression was not reduced in 3-Hz cells, 3-Hz cells incubated in KN92 continued to show significant Kv4.3 downregulation. KChIP2 expression was unaltered in the presence of KN93, excluding nonspecific effects on protein expression.
Role of Calcineurin/NFAT System
Ca2+/calmodulin also activates calcineurin, a protein phosphatase that alters gene expression by dephosphorylating the transcription factor NFAT. We first compared calcineurin activity in 1- and 3-Hz paced cells at 6 hours after pacing initiation. As shown in Figure 6A, calcineurin activity was increased >2-fold in 3-Hz paced cells. Calcineurin protein expression was not altered after 6 hours of 3-Hz pacing (Figure 6B; expanded Western blots in supplemental Figure IXC), consistent with the notion that calcineurin was functionally activated by increased Ca2+ entry in 3-Hz cells. To test for the potential role of calcineurin in Ito downregulation, Ito changes were studied in 1- and 3-Hz paced cells incubated with cyclosporin A (1 μg/mL, ≈0.8 μmol/L) or vehicle (CTL) during pacing (Ito recordings shown in Figure 6C). Cyclosporin A prevented 3-Hz pacing-induced Ito-reduction (Figure 6D), supporting the importance of calcineurin in Ito downregulation.
To assess the potential role of the calcineurin downstream mediators NFATc3 and -c4, their cellular localization was studied by confocal microscopy. Deconvolved images of NFATc3 (red) and -c4 (green) staining are shown in Figure 7A. Figure 7B shows relative nuclear/cytosolic signal ratios. Examples of ToPro3 colocalization used to identify the nuclear region are shown in supplemental Figure XI. The NFATc3 nuclear/cytosolic staining ratio was significantly increased in 3-Hz cells, compatible with nuclear relocalization. To assess nuclear NFAT localization with an independent method, we performed immunoblots on purified nuclear extracts. The results confirmed increased nuclear NFATc3 localization in 3-Hz paced cells (supplemental Figure XII). We then applied a cell-permeable NFAT inhibitor, INCA-6, to study the functional importance of NFAT in the Ito response.14 Cells were incubated with vehicle (CTL) or INCA-6 (5 μmol/L) during 24-hour pacing at 1 or 3 Hz. Ito recordings at +40 mV are shown in Figure 7C, and mean data are shown in Figure 7D. INCA-6 prevented Ito downregulation in 3-Hz cells, supporting the importance of NFAT as a mediator.
Our results point to the participation of both CaMKII and calcineurin systems as mediators of Ca2+/calmodulin effects. Previous studies suggest potential crosstalk between CaMKII and calcineurin systems.15 We wondered whether crosstalk between these systems could be contributing to calcineurin-mediated effects in our model and assessed the effects of the CaMKII inhibitor KN93 or its inactive analog KN92 on calcineurin activation by 3-Hz pacing. Calcineurin activation was suppressed by CaMKII inhibition with KN93 (Figure 6A) but not by KN92, suggesting that intact CaMKII function is needed for 3-Hz pacing-induced enhancement of calcineurin function. Further support for this notion was provided by examining the effects of CaMKII inhibition on nuclear translocation of NFATc3. As shown in Figure 7B, KN93 (but not KN92) prevented NFATc3 nuclear translocation.
In this study, we found that rapid cardiomyocyte firing decreases Ito density through downregulation of Ito α-subunit (Kv4.3) gene and protein expression. Rate-dependent Ito downregulation is mediated by increased Ca2+/calmodulin-activated CaMKII and calcineurin/NFAT signaling. Blockade of these pathways prevents rate-related Ito remodeling. A schematic summary of our findings is presented in Figure 8.
Relation to Previous Studies of Ito Downregulation in Tachypaced Models
Ventricular tachypacing is frequently used to create in vivo animal models of CHF.1,4,5 Ito downregulation is a consistent finding,1,3,5,16 generally with reductions in Ito density unaccompanied by significant changes in biophysical properties. In our in vitro tachypaced cardiomyocyte model, Ito density was similarly reduced with no change in voltage dependence or kinetic properties. As observed for in vivo tachypaced dog17,18 or rabbit19 models, Kv4.3 mRNA and protein were reduced, consistent with transcriptional downregulation.
Previous investigators have provided evidence suggesting that increased heart rate may be involved in cardiac hypertrophic signaling.20,21 The absence of changes in cellular capacitance and dimensions in 3-Hz paced cells make significant cellular hypertrophy unlikely in our model. Shortly after the onset of rapid electric stimulation (15 minutes), angiotensin II secretion and expression levels increase in cultured neonatal rat cardiomyocytes, returning to baseline after several hours.22 Incubation of epicardial ventricular myocytes with angiotensin II decreases Ito amplitude and changes its voltage-dependent and kinetic properties.23 Angiotensin receptor stimulation was not essential for Ito downregulation by 3-Hz pacing in our model, because significant downregulation continued to occur in the presence of AT1 receptor blockade with valsartan.
Ca2+/Calmodulin As a Rate Sensor
Intracellular Ca2+ concentration changes provide key signaling messages in a variety of systems.11,12,24 The role of Ca2+ is particularly important in sensing alterations in the frequency and form of neuronal activity,12 with Ca2+/calmodulin binding inducing CaMKII autophosphorylation and activation. Dynamic fluctuations in calmodulin-bound Ca2+ show both phasic components tracking intracellular Ca2+ concentration alterations and sustained changes that integrate Ca2+ concentrations over time.12 Inhibition of Ca2+ entry through L-type Ca2+ channels suppresses long-term memory effects in vivo in dogs25 and tachycardia-induced decreases in Cav1.2 protein expression in HL-1 cells.26 The importance of Ca2+/calmodulin sensing in rate-dependent Ito changes in our system was indicated by the ability of either Ca2+ channel blockade or calmodulin inhibition to prevent Ito downregulation.
Role of CaMKII and Calcineurin Signaling
Ca2+ response amplitude and duration are coupled to a variety of downstream regulatory systems, including transcription changes, for which NFAT is particularly important.24 NFAT is activated by calcineurin dephosphorylation of the NFAT regulatory domain, which is triggered by increased Ca2+/calmodulin binding.27
In vitro tachystimulation of neonatal rat cardiomyocytes28 or atrial tissue slices20 activates calcineurin/NFAT signaling. Calcineurin has been reported to alter Ito expression in a number of cardiac systems. Increased extracellular Ca2+ concentration induces Ito downregulation in rat ventricular cardiomyocytes because of reduced Kv4.2 mRNA expression, which is prevented by the calcineurin inhibitors FK506 or cyclosporin A.29 In mice, the transmural Ito gradient is set by calcineurin/NFAT-mediated downregulation of endocardial Kv4.2 and KChIP2 expression, related to higher intracellular Ca2+ concentrations in endocardium.30 Similarly, Ito and corresponding subunit mRNA downregulation resulting from acute myocardial infarction in rats is associated with increased NFAT activity and is abolished in NFATc3 knockout mice or by treatment with cyclosporin A.31 Our results agree with these studies showing that calcineurin/NFAT signaling plays a central role in Ito downregulation. In contrast, Gong et al found that overexpression of constitutively active calcineurin in neonatal rat cardiomyocytes induces hypertrophy and Ito upregulation.32 This discrepancy may be attributable to differences in the cellular environment within which calcineurin activation occurs.
High-frequency activity in neurons is transduced into CaMKII activation by increased Ca2+/calmodulin binding.12 Mice with chronic CaMKII inhibition show action potential shortening caused by upregulation of both Ito and the inward rectifier current (IK1).33 This response requires the presence of intact PLB and appears to be related to reduced CaMKII-induced PLB phosphorylation. There is evidence for interactions between calcineurin and CaMKII signaling effects on cardiac electrophysiology. Khoo et al showed that CaMKII signaling is increased in calcineurin-overexpressing mice and that their arrhythmias and left ventricular dysfunction are improved by CaMKII inhibitory drugs or in crossbred calcineurin-overexpressing/CaMKII-inhibited strains.34 We found that calcineurin activation and NFAT relocalization did not occur in cells that were tachypaced in the presence of a CaMKII inhibitor. Prior studies have shown CaMKII phosphorylation of calcineurin at very low Ca2+ concentrations that inhibit calcineurin function.15 It is conceivable that CaMKII may modulate calcineurin function by phosphorylating other regulatory proteins, but the biochemical mechanisms involved remain to be established.
Novel Findings and Potential Significance
Although tachycardia-induced cardiomyopathy consistently causes Ito downregulation in vivo,3–6 it is impossible in such a system to discriminate frequency-dependent phenomena from secondary changes attributable to altered hemodynamics, neurohormonal state, and the heart failure syndrome. Our study is, to our knowledge, the first to assess the effect of firing rate on Ito in an adult cardiomyocyte system and to study underlying regulatory mechanisms. We have uncovered a complex system in which Ca2+ acts as a frequency sensor that couples via calmodulin to downstream signals that alter Ito expression by changing the phosphorylation states of key proteins. Our results add to a growing body of evidence indicating that calcineurin/NFAT signaling acts to downregulate Ito in a variety of physiological contexts, including tachycardia, Ca2+ loading caused by increased extracellular Ca2+,29 subendocardial tissues,30 and myocardial infarction.31 Like Rossow et al,30 we found increased calcineurin activity with unchanged calcineurin expression in a context of Ca2+ loading related Ito regulation. In addition, we noted that CaMKII inhibition prevents calcineurin activity increases. Tachycardia shortens action potential duration, which would tend to reduce Ca2+ entry, as well as the time available for systole and cell contraction. Reductions in Ito might offset this effect by raising the plateau level and maintaining contraction strength. In pathological situations, however, Ito downregulation could promote arrhythmogenesis, particularly in contexts of abrupt rate slowing and reduced repolarization reserve.
There are well-recognized transmural differences in Ito density and properties.30,35 In addition, different regions of the heart vary in electrophysiological and ion current features.36 These could affect rate-dependent Ito regulation. We performed our studies in cells from the epicardium of canine left ventricles to prevent regional and transmural differences from adding uncontrolled variability to the results. A comprehensive study of the mechanisms of rate-dependent regulation of Ito in different cardiac regions and transmural layers is beyond the scope of the present study, but the issue merits further investigation. The cell-permeable agents available for CaMKII inhibition, of which KN93 is the most widely used, have imperfect specificity, including potential ICaL-blocking properties.37 For this reason, we were particularly careful to verify that the KN93 concentration we used does not block ICaL. However, we cannot totally exclude the possibility that actions of KN93 other than its CaMKII-inhibiting ability could have contributed to its effects in our system.
Tachycardia per se is clearly not the only factor that can alter Ito in ventricular tachypaced in vivo models. In addition to CHF-related neurohumoral activation and hemodynamic and metabolic changes, an altered sequence of ventricular activation can importantly alter cardiac repolarization by affecting Ito. Yu et al have shown that 2-Hz ventricular pacing of dog hearts for 3 weeks slows Ito recovery from inactivation, positively shifts inactivation voltage dependence, and reduces conductance.38 Consistent with our findings, Ito-conductance decreases were associated with comparable reductions in mRNA expression; however, Ito kinetics and voltage dependence were not changed in our cells. The discrepancies may be attributable to differences in tachypacing rate (2 versus 3 Hz) and duration (24 hours versus 3 weeks), along with the absence of altered activation pattern-related factors in our in vitro model.
NFAT is generally associated with upregulation of gene expression in hypertrophic programs, but our results implicate NFAT in tachycardia-induced Ito/Kv4.3 downregulation. Recent work has established that NFAT may also selectively repress gene expression, with important consequences for lymphocyte, adipocyte, and activity-dependent skeletal muscle gene regulation.39
We thank Chantal St-Cyr, Chantal Maltais, and Nathalie L’Heureux for technical assistance and France Thériault for secretarial help with the manuscript.
Sources of Funding
This work was supported by the Canadian Institutes of Health Research (Operating grant MOP 68929) and the Quebec Heart and Stroke Foundation.
Original received January 3, 2008; revision received July 29, 2008; accepted August 7, 2008.
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