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(Circulation Research. 1999;85:810-819.)
© 1999 American Heart Association, Inc.

Cellular Biology

Regulation of the Transient Outward K⁺ Current by Ca²⁺/Calmodulin-Dependent Protein Kinases II in Human Atrial Myocytes

Sophie Tessier, Peter Karczewski, Ernst-Georg Krause, Yves Pansard, Christophe Acar, Michel Lang-Lazdunski, Jean-Jacques Mercadier, Stéphane N. Hatem

From INSERM Unité 460 (S.T., J.-J.M., S.N.H.), Faculté de Médecine Xavier Bichat, Paris, France; Max-Delbrück Centre for Molecular Medicine (P.K., E.-G.K.), Berlin-Buch, Germany; and Service de Chirurgie Cardiaque (Y.P., C.A., M.L.-L.), Hôpital Xavier Bichat, Paris, France.

Correspondence to Pr Jean-Jacques Mercadier, INSERM Unité 460, Faculté de Médecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris, France. E-mail jjmercadier{at}wanadoo.fr

	Abstract

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Abstract—Ca²⁺/calmodulin-dependent protein kinases II (CaMKII) have important functions in regulating cardiac excitability and contractility. In the present study, we examined whether CaMKII regulated the transient outward K⁺ current (I_to) in whole-cell patch-clamped human atrial myocytes. We found that a specific CaMKII inhibitor, KN-93 (20 µmol/L), but not its inactive analog, KN-92, accelerated the inactivation of I_to (_fast: 66.9±4.4 versus 43.0±4.4 ms, n=35; P<0.0001) and inhibited its maintained component (at +60 mV, 4.9±0.4 versus 2.8±0.4 pA/pF, n=35; P<0.0001), leading to an increase in the extent of its inactivation. Similar effects were observed by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309 or with autocamtide-2–related inhibitory peptide and by external application of the calmodulin inhibitor calmidazolium, which also suppressed the effects of KN-93. Furthermore, the phosphatase inhibitor okadaic acid (500 nmol/L) slowed I_to inactivation, increased I_sus, and inhibited the effects of KN-93. Changes in [Ca²⁺]_i by dialyzing cells with 30 nmol/L Ca²⁺ or by using the fast Ca²⁺ buffer BAPTA had opposite effects on I_to. In BAPTA-loaded myocytes, I_to was less sensitive to KN-93. In myocytes from patients in chronic atrial fibrillation, characterized by a prominent I_sus, KN-93 still increased the extent of inactivation of I_to. Western blot analysis of atrial samples showed that -CaMKII expression was enhanced during chronic atrial fibrillation. In conclusion, CaMKII control the extent of inactivation of I_to in human atrial myocytes, a process that could contribute to I_to alterations observed during chronic atrial fibrillation.

Key Words: KN-93 • K⁺ channel • -CaMKII • atrial fibrillation • heart

	Introduction

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In human atrial myocytes, the transient outward K⁺ current (I_to) is essential for shaping and modulating the action potential. It is responsible for the early repolarization phase ("notch") of the action potential and for the termination of the plateau phase. In addition, the frequency-dependent reactivation of I_to explains how this current plays a major role in the adaptation of the action potential duration and cellular refractory periods to changes in cardiac cycle lengths. In both dilated and fibrillating atria, alterations of I_to, together with a marked reduction in L-type Ca²⁺ current density, are responsible for the shortening of the cellular action potential and the poor frequency-dependent adaptation of the refractory period.^{1 2} Both abnormalities favor the initiation and perpetuation of atrial arrhythmia. Studies of the mechanisms that regulate I_to are therefore crucial to understand the physiology and pathophysiology of the atrial myocardium.

The transient outward K⁺ current, recorded in isolated human atrial myocytes during a step depolarization, is composed of a rapidly inactivating component I_t and a sustained component I_sus.³ This complex time course of I_to reflects the phenotypic diversity of K⁺ channels in cardiac myocytes. Indeed, a number of electrophysiological, pharmacological, and molecular observations indicate that I_t in human atrial myocytes is the functional expression of K⁺ channels with rapid N-type inactivation, whereas I_sus is transported by slowly inactivating K⁺ channels such as hKv1.5 Shaker channels,^{4 5} which are abundantly expressed in human atrial myocardium.^{6 7} The characteristics of I_to activation and inactivation are also influenced by a variety of factors, including redox state⁸ and pharmacological agents.^{9 10} For instance, we found that the antiarrhythmic agent bertosamil can transform the noninactivating current I_sus into a rapidly inactivating current by binding to an intracellular site.¹⁰ A number of intracellular regulatory pathways can also modulate I_to in human atrial myocytes. This is the case of ß- and -adrenergic pathways, which regulate I_sus via cAMP-dependent protein kinases and protein kinases C, respectively.¹¹ It has also been proposed that the downregulation of I_to by atrial natriuretic peptide reflects the coupling between K⁺ channels and G protein.¹²

Recent studies have suggested that Ca²⁺/calmodulin-dependent protein kinases (CaMKII) modulate the inactivation of voltage-dependent K⁺ channels.^{13 14 15} For instance, CaMKII considerably slow the inactivation of Kv1.4 channels expressed in HEK-293 cells by phosphorylating a modulatory site located in the amino terminal cytoplasmic domain of these K⁺ channels. CaMKII are abundantly expressed in mammalian heart, -CaMKII being the predominant isoform.^{16 17 18 19} These kinases have important functions in regulating cardiac myocyte excitability and contractility. For instance, CaMKII modulate the frequency and voltage facilitation of L-type Ca²⁺ channels in rat ventricular myocytes²⁰ and the Ca²⁺-induced enhancement of the L-type Ca²⁺ current in rabbit ventricular myocytes.²¹ Moreover, in pathophysiological conditions characterized by [Ca²⁺]_i overload, CaMKII inhibition prevents the development of the arrhythmogenic transient inward current in rabbit ventricular myocytes.²² However, no data are available on the regulatory effect of CaMKII on K⁺ currents of cardiac myocytes, except for the identification of consensus sites for CaMKII phosphorylation on deduced amino acid sequences of several Kv channels expressed in heart.^{23 24}

The aim of the present study was to determine the contribution of CaMKII to the regulation of I_to activity in human atrial myocytes. Using whole-cell patch-clamp and immunocytochemistry techniques and various pharmacological agents, we obtained evidence that CaMKII are functionally coupled to I_to in human atrial myocytes, and that they regulate the rate and extent of inactivation of the current.

	Materials and Methods

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Cardiac Myocyte Isolation
Myocytes were enzymatically isolated from right atrial appendages obtained from 87 adult patients aged 15 to 84 years (mean 60.3±1.7 years) undergoing heart surgery for coronary artery disease (n=37), mitral valve disease (n=23), aortic valve disease (n=26), or tricuspid insufficiency (n=1). Myocytes were isolated as previously described using collagenase (type IV, Sigma) and protease (type XXIV, Sigma).¹⁰ Currents were recorded by using the patch-clamp technique in the whole-cell configuration. (Axoclamp 200A, Axon Instruments).

Solutions and Drugs
The external solution was composed of (in mmol/L) NaCl 137, KCl 5.4, CaCl₂ 2, MgCl₂ 1, HEPES 10, and glucose 10, adjusted to pH 7.3 with NaOH. For K⁺ current measurements, Na⁺ was replaced by an equimolar concentration of choline chloride, Ca²⁺ channels were blocked with 0.5 mmol/L Cd²⁺, and 10^-5 mol/L atropine was added to the external solution to prevent muscarinic receptor activation. The internal solution contained (in mmol/L) potassium aspartate 115, KCl 5, MgATP 5, sodium pyruvate 5, MgCl₂ 3, EGTA 5, and HEPES 10, adjusted to pH 7.2 with KOH. In some experiments, EGTA was replaced by 40 mmol/L BAPTA. To test the effect of Ca²⁺ on I_to the following internal solution was used (in mmol/L): KCl 115, MgATP 5, NaCl 5, MgCl₂ 3, EGTA 10, HEPES 10, and CaCl₂ 1, adjusted to pH 7.2 with KOH, which yielded a free Ca²⁺ concentration of 30 nmol/L. All experiments were carried out at room temperature (22°C to 24°C).

KN-93, KN-92, and okadaic acid were from Calbiochem. Calmidazolium was from Sigma. KN-93 was dissolved in DMSO, and the final solvent concentration was <0.05%, a concentration that had no effect on the outward K⁺ current. The Ca²⁺/calmodulin kinase II inhibitors (peptide 281 to 309 and autocamtide-2–related inhibitory peptide [AIP]; Calbiochem) were dissolved in the internal solution as well as BAPTA.

Immunoblotting and Immunohistochemistry
Ten micrograms of homogenate obtained from frozen atrial tissue was solubilized, boiled, and loaded on the top of 10% SDS polyacrylamide gels.²⁵ The membranes were processed for immunoblotting as described elsewhere.²⁶ -CaMKII were detected with an antibody that specifically recognizes the C-terminal amino acid sequence unique to a subset of -subunit variants.¹⁹ To correct for the amount of muscle protein in homogenates from individual tissue samples, the optical density (OD) values for -CaMKII were calculated relative to OD values for myosin (205 kDa) obtained from Coomassie blue–stained blot membranes.

Indirect immunofluorescence was performed on 5-µm human atrium cryosections using the anti--CaMKII antibody (5 µg/mL).

Data Analysis
The time course of I_to inactivation was best fitted by the sum of two exponential functions, I_to=Aexp(-t/_fast)+Bexp(-t/_slow)+C, where A and B are amplitude terms, t is time, _fast and _slow are time constants of the fast and slow inactivation phases, and C is the amplitude of the steady-state component. The extent of inactivation was quantified by measuring the fraction of inactivation of the outward K⁺ current defined as I_t/I_to.²⁷

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CaMKII Inhibitor KN-93 Accelerates Inactivation of Outward K⁺ Current I_to
In the majority of human atrial myocytes studied, the outward K⁺ current elicited by 10-mV incremental test pulses from -60 to +60 mV was characterized by a rapidly inactivating component (I_t) and a sustained (I_sus) component (Figure 1A). External application of KN-93 (Figure 1B), a selective inhibitor of CaMKII,²⁸ inhibited I_sus (at +60 mV, 4.9±0.4 pA/pF versus 2.8±0.4 pA/pF, in control and in KN-93 conditions, respectively; n=35, P<0.0001; Figure 1C) and increased I_t (at +60 mV, 6.5±0.5 pA/pF versus 7.3±0.5 pA/pF, in control and KN-93 conditions, respectively; n=35, P<0.01; Figure 1D), resulting in increased extent of inactivation (I_t/I_to, see Materials and Methods) of the outward K⁺ current (at +60 mV, I_t/I_to: 0.57±0.02 versus 0.74±0.02, in control and KN-93 conditions, respectively; n=35, P<0.0001). The I-V relationships showed that I_sus inhibition was significant at all potentials above +10 mV and increased with depolarization, resulting in an apparent inward rectification of I_sus (Figure 1C). In contrast, KN-93 enhanced I_t at all potentials at which this current activated and shifted its voltage dependence toward negative potentials (Figure 1D). Plotting the fast inactivation time constant (_fast, see Materials and Methods) against the test voltage showed _fast to be voltage dependent, with a mean of 188.6±19.6 ms at 0 mV and 66.9±4.4 ms at +60 mV (n=35) in control conditions (Figure 1E). Application of KN-93 decreased _fast at voltages between +20 and +60 mV (at +60 mV, _fast: 43.0±4.4 ms; n=35, P<0.0001; Figure 1E) and reduced its voltage dependence.

View larger version (22K): [in this window] [in a new window]   Figure 1. Effects of KN-93 on Ito of human atrial myocytes. Current traces elicited by 10-mV incremental 750-ms test pulses from -60 to +60 mV in control conditions (A) and in the presence of 20 µmol/L KN-93 (B). Cell capacitance: 132 pF. I-V relationships of the sustained component Isus (C) and the inactivating component It (D) in control conditions () and after the addition of 20 µmol/L KN-93 (•). Current amplitudes were normalized to cell capacitance in each cell (n=35). E, Voltage dependence of mean time constant of fast inactivation (fast) of the outward K+ current in control () and KN-93 (•) conditions. Values are mean±SEM. *P<0.05 and P<0.01; **P<0.001 and P<0.0001.

KN-93 acted slowly, as illustrated by Figure 2A, which shows that the effects of KN-93 only started to occur after 3 minutes of drug exposure, whereas steady state was obtained in 9 minutes. Washout of KN-93 (Figure 2A) was associated with a slow increase in I_t (which reached a higher amplitude than before drug application) and with a slight recovery of I_sus. These slow changes in I_to associated with KN-93 were not caused by prolonged cell dialysis or repeated membrane depolarization, as the same protocol executed with the control external solution did not cause significant changes in the characteristics of the current (not shown). Moreover, a 30-minute preincubation of rested myocytes with 20 µmol/L KN-93 also reduced I_sus (at +60 mV, 2.2±0.2 pA/pF, n=24; Figure 2B) and accelerated the rate of I_to inactivation (at +60 mV, _fast: 48.8±2.8 ms, n=24), effects similar to those of short-term application of 20 µmol/L KN-93 on I_to. These results indicate that prolonged external application of the CaMKII inhibitor KN-93 accelerated I_to inactivation.

View larger version (23K): [in this window] [in a new window]   Figure 2. Time course of the effects of KN-93. A, Normalized amplitude of It, Ito, and Isus elicited by depolarizing pulses to +50 mV from a holding potential of -60 mV, on 10-minute external application of 20 µmol/L KN-93 and during its washout. Cell capacitance: 124 pF. B, Density of Isus recorded in control conditions, on short-term KN-93 exposure and in cells pretreated for 30 minutes with 20 µmol/L KN-93. Values are mean±SEM.

Effects of KN-93 on I_to Are Largely due to Inhibition of CaMKII
We examined next whether the effects of KN-93 on I_to were related to the inhibition of CaMKII activity or to a direct effect on K⁺ channels. We first tested the effects of the functionally inactive KN-93 analog KN-92. At a concentration of 20 µmol/L, KN-92 had no significant effect on I_to (Figure 3B) compared with the outward K⁺ current elicited in control conditions (Figure 3A). A higher concentration of KN-92 (100 µmol/L) significantly inhibited I_to, an effect that predominated on I_sus (at +60 mV, 30.8±14.7%, n=8; Figure 3C), but no significant changes in the extent of inactivation were observed. Moreover, the onset of the inhibitory effect on I_sus during KN-92 exposure was rapid, being observed after the first pulse following KN-92 application; this contrasted with the slowly developing effect of KN-93 on I_to, which was still observed in myocytes pretreated with KN-92 (Figure 3D).

View larger version (31K): [in this window] [in a new window]   Figure 3. Effects of the inactive analog of KN-93, KN-92, on Ito. Family of currents elicited by 10-mV incremental test pulses from -60 to +70 mV in control conditions (A), in the presence of 20 µmol/L KN-92 (B), and in the presence of 100 µmol/L KN-92 (C). Cell capacitance: 157 pF. D, Superimposition of current traces elicited by test pulses to +50 mV from a holding potential of -60 mV recorded in control conditions, at the steady-state effect of external application of 20 µmol/L KN-92, and after external application of 20 µmol/L KN-93. Cell capacitance: 116 pF.

KN-93 inhibits CaMKII activity by blocking the binding of calmodulin to CaMKII, which is required for both the activation and the autophosphorylation of the enzyme.^{28 29} To confirm that KN-93 modulated I_to by inhibiting CaMKII, the effects of KN-93 were studied in myocytes pretreated with the calmodulin inhibitor calmidazolium. Figure 4A shows currents recorded in control conditions and during external perfusion of 50 µmol/L calmidazolium, which inhibited I_sus (at +60 mV, 4.6±0.7 pA/pF versus 3.7±0.7 pA/pF in control conditions and on 50 µmol/L calmodulin inhibitor exposure, n=14, P<0.01; Figure 4B) and increased the extent of inactivation of I_to (at +60 mV, I_t/I_to: 0.60±0.03 versus 0.67±0.03, in control conditions and on external application of calmidazolium, respectively; n=14, P<0.001). Moreover, external application of 20 µmol/L KN-93 when the steady-state effect of calmidazolium had been achieved affected neither the amplitude of the outward K⁺ current (Figure 4A) nor the I_sus density (at +60 mV, 3.4±0.7 pA/pF; n=14, not significant [NS]; Figure 4B); the extent of inactivation remained unchanged (at +60 mV, I_t/I_to: 0.68±0.03, n=14, NS).

View larger version (23K): [in this window] [in a new window]   Figure 4. Changes in Ito caused by the modulation of CaMKII activity. A, Superimposition of current traces recorded in control conditions, on 6-minute external application of 50 µmol/L calmidazolium, and on 20 µmol/L KN-93 application in the continuous presence of calmidazolium. Cell capacitance: 128 pF. B, Density of Isus recorded in control conditions, in calmidazolium conditions, and on KN-93 exposure in the presence of calmidazolium. Values are mean±SEM. *P<0.01. C, Superimposition of current traces elicited by test pulses from -60 to +50 mV obtained just after breaking the patch (control), during a 10-minute dialysis of the cell with an internal solution containing 75 µmol/L of peptide 281 to 309, and on 20 µmol/L KN-93 exposure when the steady state of dialysis of the peptide was reached. Cell capacitance: 134 pF.

Further evidence that I_to is regulated by CaMKII was obtained by dialyzing cells with a peptide corresponding to CaMKII residues 281 to 309, which is a potent calmodulin antagonist containing the calmodulin binding site of CaMKII (amino acids 290 to 309) and the autophosphorylation site (Thr²⁸⁶) of CaMKII. An example of the results is given in Figure 4C, which shows the superimposition of current traces elicited by test pulses from -60 to +50 mV, recorded just after breaking the patch and after various times of intracellular dialysis with a solution containing 75 µmol/L of the peptide. This procedure was associated with a slow fall in I_sus amplitude and an increase in the extent of current inactivation. In myocytes loaded with the peptide, external application of KN-93 had additional effects on I_to (Figure 4C), suggesting that CaMKII were only partially inhibited by the peptide. Furthermore, a sizable change in the characteristics of I_to was observed in only 5 of the 15 myocytes dialyzed with the peptide. Neither the apparently small effect nor the low success rate of experiments with peptide 281 to 309 was due to peptide lability, as AIP,³⁰ whose binding capacity cannot be altered by possible phosphorylation of Thr²⁸⁶-like peptide 281 to 309, also caused in only a limited number of cells (60%) an increase in the extent of I_to inactivation (at +60 mV, I_t/I_to: 0.52±0.05 versus 0.60±0.04, in control conditions and on internal dialysis of AIP, respectively, n=11, P<0.01) and a decrease in the amplitude of I_sus (at +60 mV, 5.2±0.7 pA/pF versus 4.4±0.6 pA/pF, n=11, P<0.01). It is therefore likely that, given the difficulty of dialyzing myocytes with high molecular weight peptides, the intracellular concentration of the latter, especially that reached in the subsarcolemmal space, may be insufficient to fully inhibit CaMKII. Consequently, in subsequent experiments, KN-93 was preferred to intracellular application of CaMKII inhibitory peptides as the most convenient tool for modulating CaMKII activity.

Inhibition of Protein Phosphatases Slows the Inactivation Kinetics of I_to
The effects of CaMKII inhibition on I_to suggested that K⁺ channels carrying the outward K⁺ current were in a phosphorylated state that could be controlled by a balance between kinase and phosphatase activities. This was tested in the next set of experiments, by studying the effect of the multifunctional phosphatase inhibitor okadaic acid on I_to. Myocytes isolated from the same right atrial samples were separated in a group of cells treated with 500 nmol/L okadaic acid for 30 minutes before starting the experiments (n=22) and a group of control cells (n=20). Figure 5A shows examples of current traces recorded in myocytes from the two groups. In okadaic acid–treated cells, the outward K⁺ current was characterized by a slight enhancement of I_sus (Figure 5B) and a lower density of I_t (at +50 mV, 6.9±0.6 pA/pF versus 5.3±0.5 pA/pF, in control and okadaic acid conditions, respectively, P<0.05; Figure 5C). The extent of inactivation of I_to was significantly decreased in the group of cells preincubated with okadaic acid compared with the control cells (at +60 mV, I_t/I_to: 0.65±0.02 versus 0.55±0.03, P<0.01). The density-voltage relationships of the two components showed that okadaic acid, in addition to its inhibitory effect on I_t, shifted I_t voltage dependence toward positive potentials (Figure 5C). Short-term external application of 500 nmol/L okadaic acid had no significant effects on I_to.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effects of okadaic acid on Ito. A, Traces of current elicited by 10-mV step depolarization from -60 to +70 mV recorded in control myocytes (C) and in myocytes pretreated with 500 nmol/L okadaic acid (OA) for 30 minutes. Cell capacitance: 93 pF and 109 pF, respectively. I-V relationships of the sustained current Isus (B) and the inactivating current It (C) in control conditions () and in cells preincubated with 500 nmol/L okadaic acid (•). Each point is the average of 20 cells in control conditions and 22 cells on okadaic acid treatment; *P<0.05. D, Effect of KN-93 on Isus recorded in control myocytes (OA-) and in myocytes preincubated with 500 nmol/L okadaic acid (OA+). Numbers inserted in bars are the numbers of cells studied. Values are mean±SEM. *P<0.01. E, Percentage of decrease of fast by KN-93 in control myocytes (OA-) and in myocytes pretreated with 500 nmol/L okadaic acid (OA+). **P<0.0001.

In cells preincubated with 500 nmol/L okadaic acid (OA+), external application of 20 µmol/L KN-93 tended to inhibit I_sus (at +60 mV, 5.7±0.7 pA/pF versus 4.1±1.1 pA/pF, with okadaic acid alone and after the addition of KN-93, respectively; Figure 5D) and decreased _fast (at +60 mV, 21.1±8.5%). However, the magnitude of the effect of KN-93 on I_to was significantly smaller in okadaic acid–treated cells (OA+) than in control cells (OA-) (Figure 5D and 5E). These results, which further demonstrate that the effects of KN-93 on I_to are due largely to the modulation of CaMKII activity, indicate that a balance between phosphatases and kinases regulates K⁺ channels in human atrial myocytes.

Modulation of I_to by Changes in [Ca²⁺]_i
The preceding results indicating a coupling between I_to and CaMKII prompted us to examine whether changes in [Ca²⁺]_i regulate the amplitude and time course of the transient outward current. This question was addressed first by using an internal solution containing 30 nmol/L of free Ca²⁺ (see Materials and Methods), a concentration that has been reported to modulate the inactivation of Kv1.4 channels.¹³ Figure 6A shows an example of the effects on I_to of dialyzing a myocyte with a Ca²⁺-containing internal solution that was associated with an enhancement of the amplitude of both I_t (at +50 mV, +28.0±3.2%, n=25, P<0.0001) and I_sus (at +50 mV, +5.1±1.7%, n=25, P<0.01). Furthermore, in myocytes dialyzed with a Ca²⁺-containing internal solution, KN-93 had a marked effect on I_to (Figure 6D and 6E). To test whether the magnitude of the effect of [Ca²⁺]_i on I_to depends on the basal activity of CaMKII, in another set of experiments, myocytes were incubated with KN-93 (20 to 40 µmol/L) for at least 30 minutes, and currents were recorded using a control external solution without the CaMKII inhibitor. Figure 6B shows an example of currents recorded in a myocyte pretreated with KN-93. Dialysis of the cell with [Ca²⁺]_i-containing internal solution caused a large increase in I_to, resulting in the apparent reversion of the effects of KN-93 on the current, particularly evident using 40 µmol/L KN-93. Statistical analysis confirmed that the effects of increasing [Ca²⁺]_i on I_to were higher in KN-93–treated than in control myocytes (at +50 mV, I_t: +35.3±4.9%, n=10, P<0.001 and I_sus: +80.0±25.4%, n=10, P<0.01). As control experiments, KN-93–pretreated myocytes were dialyzed with a control internal solution, which did not cause significant changes in I_to (n=5, data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 6. Effects on Ito of changes in free Ca2+ concentration. A, Superimposition of current traces elicited by test pulses from -60 to +50 mV recorded just after breaking the patch (control), during dialysis of the cell with a pipette solution containing 30 nmol/L of free Ca2+, and on 20 µmol/L KN-93 external application applied at the steady-state effect of Ca2+. Cell capacitance: 82 pF. B, Same procedure as in panel A applied in a cell that had been pretreated with 40 µmol/L KN-93 (control). Cell capacitance: 100 pF. C, Superimposition of current traces recorded just after breaking the patch (control), during dialysis of the cell with a pipette solution containing 40 mmol/L of the fast Ca2+ buffer BAPTA, and on application of 20 µmol/L KN-93. Cell capacitance: 122 pF. Percentage of changes of Isus (D) and It/Ito (E) caused by KN-93 (20 µmol/L) in control (open bar), calcium (filled bar), and BAPTA (hatched bar) conditions. Values are mean±SEM. *P<0.05 indicates statistical significant difference with controls.

In another set of experiments, the fast Ca²⁺ buffer BAPTA was used instead of EGTA in an attempt to reduce [Ca²⁺]_i more efficiently in the vicinity of the channels.³¹ Intracellular application of BAPTA caused an increase in the extent of current inactivation (at +60 mV, I_t/I_to: 0.57±0.03 versus 0.67±0.04, 11 of 14 myocytes; P<0.05), as illustrated by Figure 6C. Furthermore, in BAPTA-loaded myocytes, the effects of KN-93 on I_sus and on the extent of inactivation were attenuated compared with controls (at +60 mV, I_sus: 17.7±10.7%, n=7, P<0.05 and I_t/I_to: +0.5±10.0%, n=7, P<0.01; Figure 6D and 6E). Of note, the effects of calmidazolium on I_to were also reduced in cells loaded with BAPTA (data not shown, n=3). Taken together, these results indicate that I_to is modulated by changes in [Ca²⁺]_i, probably via CaMKII.

I_to Is Regulated by CaMKII in Myocytes From Fibrillating Atria
The outward K⁺ current is altered in myocytes isolated from patients with dilated or fibrillating atria,^{1 2} with a more pronounced decrease in the density of I_t than I_sus, resulting in an outward K⁺ current with a small inactivating component. Figure 7A shows a typical example of I_to recorded in myocytes from chronically fibrillating atria; note the prominent I_sus (at +60 mV, 4.5±0.4 pA/pF, n=31; NS) and reduced I_t (at +60 mV, 4.7±0.4 pA/pF, n=31; P<0.01). In these myocytes, KN-93 accelerated the rate of the outward K⁺ current inactivation (Figure 7B), resulting in an almost total suppression of the maintained current (at +60 mV, 1.7±0.2 pA/pF, n=15; P<0.0001) and restoration of a large inactivating component (at +60 mV, 6.4±0.8 pA/pF, n=15, P<0.001; Figure 7C). These results indicate that CaMKII are present in myocytes from fibrillating atria and are functionally coupled to K⁺ channels carrying I_to.

View larger version (21K): [in this window] [in a new window]   Figure 7. Effects of KN-93 on the outward K+ current recorded in myocytes isolated from fibrillating atria. Family of current traces elicited by 10-mV incremental 750-ms test pulses from -60 to +70 mV in control conditions (A) and in the presence of 20 µmol/L KN-93 (C). B, Superimposition of current traces elicited by test pulses from -60 to +50 mV in control conditions and in the presence of KN-93. Cell capacitance: 108 pF.

Increased CaMKII Expression in Fibrillating Atria
To analyze the level of CaMKII expression in human atrial myocardium, Western blot analysis was performed on proteins prepared from right atrial myocardium samples obtained with the same procedure as that used for the electrophysiological study. Figure 8A shows the Western blot obtained with the -CaMKII–specific antibody in atrial samples from patients listed in the Table. -CaMKII was detected in all the samples, but densitometric analysis showed that its expression was significantly enhanced from 5.9±1.0 OD (n=7) in control atria to 11.2±2.1 OD (n=5) in chronically fibrillating atria (Figure 8B; P=0.032). Immunocytochemical analysis of tissue sections with the same -CaMKII–specific antibody showed that specific staining predominated in atrial myocytes. The -CaMKII appeared to be located throughout the cell body, but more intense staining was observed in intercalated disks (Figure 9A), which contain most Kv1.5 channels.⁷ As a negative control, the primary antibody was preincubated with an excess of antigen, leading to the absence of specific staining (Figure 9B). A similar expression pattern was observed in tissue sections obtained from chronically fibrillating atria.

View larger version (23K): [in this window] [in a new window]   Figure 8. -CaMKII protein expression in human atrial myocardium. A, Immunoblot of homogenates of atrial samples from control patients (control) and from patients with chronic atrial fibrillation (AF). B, Statistical evaluation of data obtained by densitometry of the immunoblot shown in panel A. Columns represent mean±SEM. *P<0.05. For experimental details, see Materials and Methods.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of the Patients

View larger version (73K): [in this window] [in a new window]   Figure 9. A, Immunolocalization of -CaMKII in human atrial myocardium showing both a striated staining inside the cell and a staining at the periphery (predominantly at the level of the intercalated disks; see arrow). B, Negative immunostaining control performed in the presence of the peptide used for the generation of the primary antibody. Bar=40 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
To our knowledge, this is the first demonstration that I_to is regulated by CaMKII in cardiac myocytes. Our results also suggest that upregulation of these protein kinases could contribute to the electrical remodeling of diseased atrial myocardium.

The conclusion that CaMKII regulate I_to is based on a strong body of evidence. KN-93, a specific inhibitor of CaMKII, but not its functionally inactive analog KN-92, had a marked effect on I_to. At a high concentration (100 µmol/L), KN-92 caused a use-dependent inhibition of I_to, pointing to direct binding of this molecule to K⁺ channels, an effect that may also be shared by KN-93 and may explain the increase in I_t after drug washout (Figure 2A). In addition, in keeping with the mechanism of action of KN-93 (which blocks the calmodulin binding to CaMKII),^{28 29} the calmodulin inhibitor calmidazolium suppressed the effects of KN-93 on I_to. Calmodulin inhibition was associated with changes in I_to similar to those observed with the CaMKII inhibitor, suggesting that the two compounds modulate a common regulatory pathway. Intracellular dialysis of a synthetic peptide inhibitor of CaMKII containing the calmodulin binding site (amino acids 290 to 309) and the autophosphorylation site (Thr²⁸⁶) of CaMKII or with AIP, a more stable peptide than the former, had effects on I_to similar to those of external KN-93 application, ie, an increased extent and accelerated rate of inactivation of I_to. The low success rate in experiments with both peptides, as well as their weaker effects on I_to relative to those of KN-93, were likely due to the difficulty in dialyzing the subsarcolemmal region of the cells with such large molecules.³² Moreover, CaMKII may be tightly associated with K⁺ channels, as is the case for the N-methyl-D-aspartate receptor,³³ explaining the poor accessibility of the enzymes. The finding that phosphatase inhibition by okadaic acid altered the extent of inactivation of I_to also indicates that the activity of K⁺ channels carrying I_to depends on their phosphorylation state. Given that pretreatment of myocytes with okadaic acid attenuated the effects of KN-93 on I_to, CaMKII probably contribute to the tonic phosphorylation of K⁺ channels. Finally, cell dialysis with BAPTA, which buffers Ca²⁺ in the subsarcolemmal space more efficiently than EGTA, also modified the rate and extent of I_to inactivation, indicating that the time course of the current is controlled by [Ca²⁺]_i-dependent processes. Because (1) the effects of BAPTA on I_to resemble those of KN-93, calmidazolium, or CaMKII inhibitory peptides and (2) the sensitivity of I_to for KN-93 is reduced in myocytes dialyzed with BAPTA, the effects of changes in [Ca²⁺]_i on I_to may be largely mediated by CaMKII. The observation that increasing [Ca²⁺]_i had a limited effect on I_to, which in these conditions became exquisitely sensitive to KN-93, suggests that in human atrial myocytes and/or in our experimental conditions, CaMKII may already be activated. Indeed, pretreating myocytes with KN-93 to inhibit CaMKII enhances the effects of increasing [Ca²⁺]_i on I_to. The most likely explanation for this finding is that increasing [Ca²⁺]_i causes an excess in Ca²⁺ calmodulin, which is able to recruit and activate CaMKII, probably in a competitive fashion against KN-93 in keeping with the mechanism of action of this compound.²⁸

Although the present results point to Ca²⁺-dependent regulation of I_to mainly via CaMKII activation, they do not rule out the possibility that part of the effects of KN-93 on the current are due to direct effects of the compound on K⁺ channels, distinct from those shared with its inactive analog KN-92. For instance, in rabbit ventricular myocytes, the peak transient outward current is blocked significantly by KN-93 but not by the inactive analog KN-92 or by a CaMKII inhibitory peptide.³⁴ In human atrial myocytes, such direct blockade of I_to by KN-93 could explain the additional effect of the compound in other experimental conditions in which CaMKII was inhibited (Figures 4 and 6).

Inhibition of CaMKII markedly accelerated the rate of current inactivation, resulting in a prominent I_t with a shift toward negative potentials in its density-voltage relationships and a reduced I_sus associated with inward rectification. These effects suggest that CaMKII inhibition alters the gating characteristics of channels carrying the outward K⁺ current, resulting in an increased fraction of current that inactivates. Voltage-gated K⁺ channels, which are thought to carry the outward K⁺ current in cardiac myocytes, inactivate via two mechanisms: rapid N-type inactivation, which is described by a "ball-and-chain" model, and slow C-type inactivation.³⁵ Both mechanisms are modulated by several factors,³⁶ including serine/threonine phosphorylation processes.³⁷ The presence of consensus sites for CaMKII³⁸ on deduced amino acid sequences of Kv1.5 channels,⁶ the main molecular basis for I_sus in human atrial myocytes,^{4 5} is consistent with the possibility of direct phosphorylation of these channels by CaMKII, which may modulate its rate of inactivation.¹³ The inactivation of K⁺ channels can also be markedly accelerated by coexpression of auxiliary cytoplasmic ß subunits with pore-forming subunits of Kv1 channels, conferring rapid inactivation to noninactivating delayed rectifier currents.^{39 40} Interestingly, the interaction between and ß subunits of Kv channels is also regulated by second messengers, including cAMP-dependent protein kinases (PKA) and protein kinases C, which modulate the extent of ß-current inactivation.^{27 41} It has been reported that PKA also alter Kvß1.3 subunit–mediated inactivation of Kv1.5 channels, resulting in a current with a reduced extent and rate of inactivation.⁴² Our results do not allow us to draw firm conclusions on the mechanism by which CaMKII regulate the inactivation of K⁺ channels carrying I_to. Nevertheless, it is interesting to note that the effects of CaMKII inhibition on I_to share certain features with those of hKvß1.3 subunits on hKv1.5 K⁺ channels, which are expressed in human atrial myocardium, ie, partial inactivation and inward rectification with depolarization⁴⁰ ; as with PKA,⁴² the interaction between the two subunits may be regulated by CaMKII.

It is already known that CaMKII regulate the inactivation of K⁺ channels carrying voltage-dependent outward K⁺ current in neurons,¹³ photoreceptor cells,¹⁴ and murine colonic myocytes.¹⁵ Indeed, the frequency-dependent inactivation of the K⁺ current carried by Shaker Kv1.4 is regulated by CaMKII in a manner somewhat similar to the effects of these kinases on the outward K⁺ current of human atrial myocytes.¹³ In this latter study, increasing the [Ca²⁺]_i or inhibiting phosphatases with okadaic acid drastically slowed the inactivation of the Kv1.4 current, which was accelerated when CaMKII were inhibited by KN-93. Taken together, these studies suggest that CaMKII are involved in controlling repolarization in excitable cells. In human atrial myocytes, the fall in the rate of I_to inactivation caused by CaMKII should enhance the maintained level of the outward K⁺ current within a large range of potentials and thus shorten the plateau phase of the action potential. As a result, Ca²⁺ influx through L-type Ca²⁺ channels and, in turn, Ca²⁺ release from the sarcoplasmic reticulum could be reduced, thereby preventing [Ca²⁺]_i accumulation and further activation of CaMKII. Our observation, in chronically fibrillating atrial myocardium, of upregulated expression of -CaMKII, which appears to be functional and coupled to K⁺ channels, raises questions as to the contribution of this regulatory process to the electrical remodeling that occurs during atrial fibrillation. Indeed, there is evidence that changes in [Ca²⁺]_i homeostasis may initiate electrical remodeling during atrial fibrillation, which is characterized by a marked shortening of the action potential plateau phase.^{43 44 45} It is tempting to speculate that the upregulation of CaMKII during atrial fibrillation, by reducing the extent of inactivation of I_to, reduces Ca²⁺ influx and therefore minimizes Ca²⁺ overload. As CaMKII are sensitive to the rate of [Ca²⁺]_i oscillations that they can decode into distinct amounts of kinase activity,⁴⁶ it is also possible that the coupling between CaMKII and K⁺ channels may contribute to the adaptation of the electrical activity of human atrial myocardium to sustained changes in heart rate, such as those occurring during chronic atrial arrhythmia.

	Acknowledgments

 
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), Association Française contre les Myopathies (AFM), Fondation pour la Recherche Médicale (FRM), and Assistance Publique-Hôpitaux de Paris (AOB94038). Sophie Tessier was supported by a grant from Ministère de l'Enseignement Supérieur et de la Recherche. We thank Josette Riou for assistance with the collection of atrial samples. We are indebted to Dr Jane-Lyse Samuel for help and advice with the immunostaining. This manuscript is dedicated to the memory of Prof Edouard Coraboeuf.

Received March 4, 1999; accepted July 26, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Le Grand B, Hatem S, Deroubaix E, Couetil J-P, Coraboeuf E. Depressed transient outward and calcium currents in dilated human atria. Cardiovasc Res. 1994;28:548–556.[Medline] [Order article via Infotrieve]

2. Van Wagoner DR, Pond AL, McCarthy PM, Trimmer JS, Nerbonne JM. Outward K⁺ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res. 1997;80:772–781.[Abstract/Free Full Text]

3. Escande D, Coulombe A, Faivre J-F, Deroubaix E, Coraboeuf E. Two types of transient outward currents in adult human atrial cells. Am J Physiol. 1987;252:H142–H148.[Abstract/Free Full Text]

4. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes. Evidence for a novel delayed rectifier K⁺ current similar to Kv1.5 cloned channel currents. Circ Res. 1993;73:1061–1076.[Abstract/Free Full Text]

5. Fedida D, Wible B, Wang Z, Fermini B, Faust F, Nattel S, Brown AM. Identity of a novel delayed rectifier current from human heart with a cloned K⁺ channel current. Circ Res. 1993;73:210–216.[Abstract]

6. Tamkun MM, Knoth KM, Walbridge JA, Kroemer H, Roden DM, Glover DM. Molecular cloning and characterization of two voltage-gated K⁺ channel cDNAs from human ventricle. FASEB J. 1991;5:331–337.[Abstract]

7. Mays DJ, Foose JM, Philipson LH, Tamkun MM. Localization of the Kv1.5 K⁺ channel protein in explanted cardiac tissue. J Clin Invest. 1995;96:282–292.

8. Ogbaghebriel A, Shrier A. Inhibition of metabolism abolishes transient outward current in rabbit atrial myocytes. Am J Physiol. 1994;266:H182–H190.[Abstract/Free Full Text]

9. Dukes ID, Morad M. Tedisamil inactivates transient outward K⁺ current in rat ventricular myocytes. Am J Physiol. 1989;257:H1746–H1749.[Abstract/Free Full Text]

10. Tessier S, Rücker-Martin C, Macé L, Coraboeuf E, Mercadier J-J, Hatem SN. The antiarrhythmic agent bertosamil induces inactivation of the sustained outward K⁺ current in human atrial myocytes. Br J Pharmacol. 1997;122:291–301.[Medline] [Order article via Infotrieve]

11. Li G-R, Feng J, Wang Z, Fermini B, Nattel S. Adrenergic modulation of ultrarapid delayed rectifier K⁺ current in human atrial myocytes. Circ Res. 1996;78:903–915.[Abstract/Free Full Text]

12. Le Grand B, Deroubaix E, Couétil J-P, Coraboeuf E. Effects of atrionatriuretic factor on Ca²⁺ current and Ca_i-independent transient outward K⁺ current in human atrial cells. Pflügers Arch. 1992;421:486–491.[Medline] [Order article via Infotrieve]

13. Roeper J, Lorra C, Pongs O. Frequency-dependent inactivation of mammalian A-type K⁺ channel Kv1.4 regulated by Ca²⁺/calmodulin-dependent protein kinase. J Neurosci. 1997;17:3379–3391.[Abstract/Free Full Text]

14. Peretz A, Abitbol I, Sobko A, Wu C-F, Attali B. A Ca²⁺/calmodulin-dependent protein kinase modulates Drosophila photoreceptor K⁺ currents: a role in shaping the photoreceptor potential. J Neurosci. 1998;18:9153–9162.[Abstract/Free Full Text]

15. Koh SD, Perrino BA, Hatton WJ, Kenyon JL, Sanders KM. Novel regulation of the A-type K⁺ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II. J Physiol (Lond). 1999;517:75–84.[Abstract/Free Full Text]

16. Tobimatsu T, Fujisawa H. Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem. 1989;264:17907–17912.[Abstract/Free Full Text]

17. Baltas LG, Karczewski P, Krause E-G. The cardiac sarcoplasmic reticulum phospholamban kinase is a distinct -CaM kinase isozyme. FEBS Lett. 1995;373:71–75.[Medline] [Order article via Infotrieve]

18. Singer HA, Benscoter HA, Schworer CM. Novel Ca²⁺/calmodulin-dependent protein kinase II -subunit variants expressed in vascular smooth muscle, brain, and cardiomyocytes. J Biol Chem. 1997;272:9393–9400.[Abstract/Free Full Text]

19. Hoch B, Haase H, Schulze W, Hagemann D, Morano I, Krause E-G, Karczewski P. Differentiation-dependent expression of cardiac -CaMKII isoforms. J Cell Biochem. 1998;68:259–268.[Medline] [Order article via Infotrieve]

20. Xiao R-P, Cheng H, Lederer WJ, Suzuki T, Lakatta EG. Dual regulation of Ca²⁺/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci U S A. 1994;91:9659–9663.[Abstract/Free Full Text]

21. Anderson ME, Braun A, Schulman H, Premack BA. Multifunctional Ca²⁺/calmodulin-dependent protein kinase mediates Ca²⁺-induced enhancement of the L-type Ca²⁺ current in rabbit ventricular myocytes. Circ Res. 1994;75:854–861.[Abstract/Free Full Text]

22. Wu Y, Roden DM, Anderson ME. Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current. Circ Res. 1999;84:906–912.[Abstract/Free Full Text]

23. Roberds SL, Tamkun MM. Cloning and tissue-specific expression of five voltage-gated potassium channel cDNAs expressed in rat heart. Proc Natl Acad Sci U S A. 1991;88:1798–1802.[Abstract/Free Full Text]

24. Attali B, Lesage F, Ziliani P, Guillemare E, Honoré E, Waldmann R, Hugnot J-P, Mattéi M-G, Lazdunski M, Barhanin J. Multiple mRNA isoforms encoding the mouse cardiac Kv1–5 delayed rectifier K⁺ channel. J Biol Chem. 1993;268:24283–24289.[Abstract/Free Full Text]

25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685.[Medline] [Order article via Infotrieve]

26. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Natl Acad Sci U S A. 1979;76:4350–4354.[Abstract/Free Full Text]

27. Levin G, Chikvashvili D, Singer-Lahat D, Peretz T, Thornhill WB, Lotan I. Phosphorylation of a K⁺ channel subunit modulates the inactivation conferred by a ß subunit. Involvement of cytoskeleton. J Biol Chem. 1996;271:29321–29328.[Abstract/Free Full Text]

28. Sumi M, Kiuchi K, Ishikawa T, Ishii A, Hagiwara M, Nagatsu T, Hidaka H. The newly synthesized selective Ca²⁺/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. Biochem Biophys Res Comm. 1991;181:968–975.[Medline] [Order article via Infotrieve]

29. Rich RC, Schulman H. Substrate-directed function of calmodulin in autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II. J Biol Chem. 1998;273:28424–28429.[Abstract/Free Full Text]

30. Ishida A, Kameshita I, Okuno S, Kitani T, Fujisawa H. A novel highly specific and potent inhibitor of calmodulin-dependent protein kinase II. Biochem Biophys Res Commun. 1995;212:806–812.[Medline] [Order article via Infotrieve]

31. Stern MD. Buffering of calcium in the vicinity of a channel pore. Cell Calcium. 1992;13–183-192.

32. McCarron JG, McGeown JG, Reardon S, Ikebe M, Fay FS, Walsh JV Jr. Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II. Nature. 1992;357:74–77.[Medline] [Order article via Infotrieve]

33. Soren Leonard A, Lim IA, Hemsworth DE, Horne MC, Hell JW. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-D-aspartate receptor. Proc Natl Acad Sci U S A. 1999;96:3239–3244.[Abstract/Free Full Text]

34. Anderson ME, Braun AP, Wu Y, Lu T, Wu Y, Schulman H, Sung RJ. KN-93, an inhibitor of multifunctional Ca⁺⁺/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart. J Pharmacol Exp Ther. 1998;287:996–1006.[Abstract/Free Full Text]

35. Rasmusson RL, Morales MJ, Wang S, Liu S, Campbell DL, Brahmajothi MV, Strauss HC. Inactivation of voltage-gated cardiac K⁺ channels. Circ Res. 1998;82:739–750.[Abstract/Free Full Text]

36. Baukrowitz T, Yellen G. Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K⁺ channel. Science. 1996;271:653–656.[Abstract]

37. Kupper J, Bowlby MR, Marom S, Levitan IB. Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel. Pflügers Arch. 1995;430:1–11.

38. Pearson RB, Kemp BE. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81.[Medline] [Order article via Infotrieve]

39. Majumder K, De Biasi M, Wang Z, Wible BA. Molecular cloning and functional expression of a novel potassium channel ß-subunit from human atrium. FEBS Lett. 1995;361:13–16.[Medline] [Order article via Infotrieve]

40. England SK, Uebele VN, Kodali J, Bennett PB, Tamkun MM. A novel K⁺ channel ß-subunit (hKvß1.3) is produced via alternative mRNA splicing. J Biol Chem. 1995;270:28531–28534.[Abstract/Free Full Text]

41. Levy M, Jing J, Chikvashvili D, Thornhill WB, Lotan I. Activation of a metabotropic glutamate receptor and protein kinase C reduce the extent of inactivation of the K⁺ channel Kv1.1/Kvß1.1 via dephosphorylation of Kv1.1. J Biol Chem. 1998;273:6495–6502.[Abstract/Free Full Text]

42. Kwak Y-G, Hu NN, Wei J, George AL, Grobaski TD, Tamkun MM, Murray KT. Protein kinase A phosphorylation alters Kvß1.3 subunit-mediated inactivation of the Kv1.5 potassium channel. J Biol Chem. 1999;274:13928–13932.[Abstract/Free Full Text]

43. Goette A, Honeycutt C, Langberg JJ. Electrical remodeling in atrial fibrillation. Time course and mechanisms. Circulation. 1996;94:2968–2974.[Abstract/Free Full Text]

44. Leistad E, Aksnes G, Verburg E, Christensen G. Atrial contractile dysfunction after short-term atrial fibrillation is reduced by verapamil but increased by Bay K8644. Circulation. 1996;93:1747–1754.[Abstract/Free Full Text]

45. Dispersyn G, Duimel H, Ausma J, Borgers M. Ultrastructural calcium distribution in cardiomyocytes after chronic atrial fibrillation. Circulation. 1998;98(suppl I):I-683. Abstract 3592.

46. De Koninck P, Schulman H. Sensitivity of CaM kinase II to the frequency of Ca²⁺ oscillations. Science. 1998;279:227–230.[Abstract/Free Full Text]

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