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(Circulation Research. 1999;84:906-912.)
© 1999 American Heart Association, Inc.

Original Contribution

Calmodulin Kinase Inhibition Prevents Development of the Arrhythmogenic Transient Inward Current

Yuejin Wu, Dan M. Roden, Mark E. Anderson

From the Departments of Medicine (Y.W., D.M.R., M.E.A.) and Pharmacology (D.M.R., M.E.A.), Vanderbilt University, Nashville, Tenn.

Correspondence to Mark Anderson, Vanderbilt University Medical Center, 315 Medical Research Building II, Nashville, TN 37232-6300. E-mail mark.anderson{at}mcmail.vanderbilt.edu

	Abstract

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Abstract—Although it is widely accepted that afterdepolarizations initiate arrhythmias when action potentials are prolonged, the underlying mechanisms are unclear. In this study, we tested the hypothesis that action potential prolongation would raise intracellular calcium and thereby activate the arrhythmogenic transient inward current (I_ti). Furthermore, given that I_ti can be activated by sarcoplasmic reticulum Ca²⁺ release, we tested the hypothesis that inhibition of calmodulin (CaM) kinase would prevent I_ti. Isolated rabbit ventricular myocytes were studied with whole-cell–mode voltage clamp. Stimulation with a prolonged action potential clamp, under near-physiological conditions, increased [Ca²⁺]_i. I_ti was reproducibly induced in 60 of 60 cells, but I_ti was not seen with the use of a shorter action potential waveform (n=12). I_ti was associated with a secondary elevation in [Ca²⁺]_i. When [Ca²⁺]_i buffering was enhanced by dialysis with BAPTA (20 mmol/L, n=9), no I_ti was present. The Na⁺/Ca²⁺ exchanger was likely responsible for I_ti, because I_ti was inhibited by the Na⁺/Ca²⁺ exchanger inhibitory peptide XIP (10 µmol/L, n=6), but not by an inactive scrambled peptide (10 µmol/L, n=5) or by the Cl^– current antagonist niflumic acid (10 to 40 µmol/L, n=9). Activator Ca²⁺ from the sarcoplasmic reticulum was essential for development of I_ti, because it was prevented by pretreatment with ryanodine (10 µmol/L, n=6) or thapsigargin (1 µmol/L, n=6). Two different CaM kinase inhibitory peptides (n=16) and a CaM inhibitory peptide (n=4) completely suppressed I_ti. These results are consistent with the hypothesis that CaM kinase plays a role in arrhythmias related to increased [Ca²⁺]_i.

Key Words: calmodulin kinase • sarcoplasmic reticulum • transient inward current • Ca²⁺-activated chloride current • Na⁺/Ca²⁺ exchanger

	Introduction

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Calmodulin (CaM) kinase is a ubiquitous serine/threonine kinase that has recently been shown to modulate the release of activator Ca²⁺ during excitation-contraction coupling in ventricular myocytes.¹ CaM kinase is activated by increased [Ca²⁺]_i² ; once activated by Ca²⁺ bound to CaM, CaM kinase can undergo a series of autophosphorylation events to become Ca²⁺ independent.³ We have recently found that this Ca²⁺-independent component of CaM kinase activity increases during action potential prolongation and early afterdepolarizations (EADs) in isolated rabbit ventricle.⁴ CaM kinase acts at key sites for [Ca²⁺]_i homeostasis and is known to increase L-type Ca²⁺ current^{5 6 7} and facilitate the uptake⁸ and release^{1 9} of Ca²⁺ by the sarcoplasmic reticulum (SR) in ventricular myocytes. Thus, CaM kinase activity could be important for the development of [Ca²⁺]_i overload arrhythmias.

One mechanism for arrhythmias due to [Ca²⁺]_i overload is activation of a transient inward current (I_ti).¹⁰ In non–voltage-clamped cells I_ti causes cell membrane depolarization resulting in delayed afterdepolarizations (DADs) and triggered arrhythmias.¹¹ Under near-physiological conditions, I_ti may be due to the electrogenic Na⁺/Ca²⁺ exchanger operating in forward mode,¹² but results of other experiments performed under less physiological conditions (eg, in the absence of extracellular Na⁺ or after Na⁺/K⁺ ATPase inhibition) suggest that this current could also be due to a Ca²⁺-activated cation nonselective current (I_CAN)^{12 13 14 15 16} or a Ca²⁺-activated Cl^– current (I_Ca/Cl).^{16 17 18 19}

Many conditions such as ischemia and tachycardia²⁰ may be associated with increased [Ca²⁺]_i. Prolongation of action potential repolarization, a therapeutic action of a variety of antiarrhythmic agents,²¹ is also associated with arrhythmias in cardiomyopathy²² and congenital long-QT syndromes. Whereas the ionic mechanisms underlying action potential prolongation in these settings are becoming better understood, those underlying the accompanying afterdepolarizations and triggered arrhythmias are less well understood. In these studies, we present evidence that the elevated [Ca²⁺]_i, which follows action potential prolongation, can activate I_ti due to Na⁺/Ca²⁺ exchanger activity and that this activation is dependent on CaM kinase. These results suggest that this CaM kinase–dependent mechanism may trigger arrhythmias arising when action potentials are prolonged.

	Materials and Methods

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Preparation of Isolated Ventricular Myocytes
Isolation of rabbit ventricular myocytes was performed as previously described.⁵ New Zealand White rabbits of either sex (2 to 3 kg) were euthanized with a pentobarbital overdose (50 mg/kg, IV) after heparin infusion (150 U/kg, IV). The collagenase-containing solution was prepared in nominally Ca²⁺-free solution containing 60 U/mL collagenase type I from Worthington Biochemicals and 0.1 U/mL type XIV protease from Sigma. Myocytes were dispersed by gentle agitation and then maintained in standard saline solution containing 1.8 mmol/L CaCl₂. Myocytes were studied within 8 hours after dispersion. Only rod-shaped quiescent myocytes with clear striations from the left ventricle were studied.

Solutions
The solutions for preparation of isolated ventricular myocytes were described previously.⁵ The bath solution contained (in mmol/L) NaCl 140.0, HEPES 5.0, glucose 10.0, KCl 5.4, CaCl₂ 2.5, and MgCl₂ 1.0. The pH was adjusted to 7.4 with 10 N NaOH. The intracellular solution contained (in mmol/L) potassium aspartate 120.0, HEPES 5.0, KCl 25.0, disodium ATP 4.0, MgCl₂ 1.0, disodium phosphocreatine 2.0, and sodium GTP 2.0. The pH was adjusted to 7.2 with 1 N KOH. In some experiments, the Cl^– current antagonist niflumic acid²³ was added to the bath solution for a final concentration of 10 to 40 µmol/L. The SR Ca²⁺ release channel blocker ryanodine (10 µmol/L) or the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase antagonist thapsigargin (1 µmol/L, Calbiochem) were included in other experiments. Thapsigargin was added as a DMSO stock solution with a final DMSO concentration of 0.0001 vol %. Unless otherwise noted, all chemicals were from Sigma.

Electrophysiology
Current Clamp
Cells were stimulated at 0.1 Hz in current clamp mode with 1.0- to 2.0-nA pulses of depolarizing current (1.25 times threshold) for 3 to 4 ms at room temperature (20°C to 23°C). Action potentials were low pass filtered at 2 kHz and sampled at 2.5 kHz with a 12-bit analog to digital converter (Digidata 1200 B) from Axon Instruments. Representative short (APD₅₀=194 ms; APD₉₀=217 ms) and long (APD₅₀=422; APD₉₀=561 ms) action potential waveforms were digitized and stored for application as voltage commands for most experiments using pClamp 6.03 (Figure 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Effect of stimulation with a long and short action potential waveform. A, Stimulation with a long action potential waveform resulted in Iti after completion of repolarization, as indicated (arrow). Outward deflection at the terminal portion of the action potential clamp was likely the inward rectifier K+ current as it was abolished by 0.5 mmol/L Ba2+ (not shown). B, Stimulation with a short action potential waveform failed to elicit Iti. An inward current was present coincidentally with terminal repolarization using the short, but not the long, action potential waveform and was likely the result of repolarization-associated forward-mode Na+/Ca2+ exchanger activity, as described by other investigators.12 39 Both waveforms were applied at 0.5 Hz. C, The steady-state [Ca2+]i transient was greater during stimulation with the long than with the short AP waveform.

Voltage Clamp
Isolated myocytes were studied with whole-cell–mode voltage-clamp configuration at 32°C on a heated stage (Warner Instrument Corp) using an amplifier (Axopatch 200B, Axon Instruments), as previously described.^{4 5} Micropipette electrode resistance was 1.5 to 3.5 M when micropipettes were filled with the intracellular solution. Membrane currents were low pass filtered at 2 kHz and sampled at 6.7 kHz. The digitized signals were stored for later analysis with pClamp 6.03 (Axon Instruments) and Sigma Plot (Jandel Scientific). Cell membrane capacitance (167±2.2 pF) was measured using the integral of the current transient after a 10-mV hyperpolarizing step from a holding potential of –80 mV or a 10-mV depolarizing step from a holding potential of 0 mV.

I_ti Measurement
After a dialysis-and-stabilization period of 5 minutes, cells were repetitively (50 times) depolarized at 0.5 Hz with either a long or a short action potential command waveform (Figure 1). The presence or absence of oscillatory currents after completion of repolarization (I_ti) was noted during the first 50 depolarizations, and peak I_ti was measured as the difference between the current at the onset of the oscillation and the current at maximum excursion of the oscillation. Oscillatory currents were also induced at different test potentials following conventional conditioning steps (0.5 Hz) from –80 to +10 or +30 mV (Figure 2D).

View larger version (22K): [in this window] [in a new window]   Figure 2. Iti is likely due to the Na+/Ca2+ exchanger. A through C, Data tracings from separate myocytes stimulated with the long action potential waveform (Figure 1A). A, Tracing from a myocyte dialyzed with XIP, which prevented Iti. B, Inactive control peptide sXIP did not prevent Iti. C, Niflumic acid did not suppress Iti. D, Schematic depiction of conditioning and test command voltages, applied at 0.5 Hz, used to generate the raw data tracings (E) and current-voltage relationships (F). E, Oscillatory currents (single arrows) in response to a range of test cell membrane potential (Vm) steps with Vm indicated next to each tracing. The large inward current present at the –80-mV test Vm was the inward rectifier, because it was abolished by addition of Ba2+ (0.5 mmol/L) (not shown). A small oscillatory biphasic current was present in later conditioning steps (double arrows) and was absent in XIP-treated cells. Erev for the oscillatory currents was not changed by reversing the order of test Vm commands (not shown). F, Current-voltage relationship of the oscillatory currents. Oscillatory inward currents were prevented by dialysis with XIP. XIP significantly reduced peak currents (P0.005) at all test potentials except for –80 mV (P=0.06) and +60 mV (P=0.059) relative to control. Niflumic acid did not affect the inward currents but significantly reduced the outward current at +40 (P=0.003) and +60 mV (P=0.026) compared with control.

[Ca²⁺]_i Measurements
Fluo 3
[Ca²⁺]_i was monitored to compare the effect of long and short action potential waveforms on [Ca²⁺]_i by including the pentapotassium salt of the fluorescent Ca²⁺ indicator fluo 3 (Molecular Probes) in the pipette solution (100 µmol/L) as previously described, with minor modifications.⁵ Voltage signals were low pass filtered at 50 Hz before analysis. Steady-state fluo 3 [Ca²⁺]_i transients were integrated using pClamp 6.03.

Indo 1
The pentapotassium salt of the ratiometric Ca²⁺ indicator indo 1 (100 to 300 µmol/L) was dialyzed into cells to verify a secondary increase in [Ca²⁺]_i during I_ti. Excitation light was band pass filtered (360±20 nm) and reflected with a dichroic mirror (375 nm, Omega Optical) to the myocyte on the inverted microscope stage (Nikon). Emission light (ie, >375 nm) was reflected through an adjustable window to exclude extraneous fluorescence, split with a second dichroic mirror (455 nm), and represented as the ratio of 405±20/500±20 nm. Photobleaching was limited to 10% of peak output by an electronically controlled shutter device (Uniblitz) for both indo 1 and fluo 3 experiments.

Inhibitory Peptides
In separate experiments, cells were dialyzed for 5 minutes with inhibitory peptides against CaM kinase, protein kinase A (PKA), and protein kinase C (PKC) before the experimental protocol was initiated. CaM kinase was inhibited with the following peptides (20 µmol/L): 273-302, 291-317, and AC3-I (KKALHRQEAVDCL).²⁴ The inhibitory peptide 273-302 is modeled after the autoinhibitory region of CaM kinase and competitively binds to the catalytic site, whereas peptide 291-317 is modeled after the CaM binding region and so competitively inhibits CaM kinase and other CaM-dependent processes.^{2 5} The amino acid sequence HRQEAVDC in AC3-I corresponds to the surrounding autophosphorylation site on CaM kinase, except that T (Thr286/287) is modified to A (Ala) to prevent phosphorylation. Thus, AC3-I is a modified CaM kinase substrate that competitively inhibits (in vitro IC₅₀, 3 µmol/L) interaction of activated CaM kinase with substrate.²⁴ Other serine threonine kinases are not significantly inhibited by AC3-I at the concentration used in these experiments.²⁴ The peptide AC3-C (KKALHAQERVDCL) has no inhibitory activity²⁴ and was included in the pipette solution (20 µmol/L) in control experiments. CaM kinase inhibitory peptides were prepared using a solid-phase peptide synthesizer (Applied Biosystems) and purified by reverse-phase HPLC. The sequences were confirmed by automated sequencing. These peptides were generous gifts of Dr Howard Schulman (Stanford University, Stanford, Calif).

The competitive PKA inhibitory peptide (IC₅₀=0.2 µmol/L) corresponds to positions 6 to 22 of PKA and was included in the pipette solution at 10 µmol/L. The competitive PKC inhibitory peptide (IC₅₀=0.3 µmol/L) corresponds to the autoinhibitory domain positions 19 to 36 of PKC and was included in the pipette solution at 20 µmol/L. Both PKA and PKC inhibitory peptides were obtained commercially (Gibco-BRL). The Na⁺/Ca²⁺ exchanger inhibitory peptide (XIP) is modeled after a putative CaM binding site on the Na⁺/Ca²⁺ exchanger and acts to potently inhibit Na⁺/Ca²⁺ exchanger current in ventricular myocytes when included in the dialysate (10 or 20 µmol/L).²⁵ Scrambled XIP (sXIP) does not affect the Na⁺/Ca²⁺ exchanger and was included in the pipette solution (10 µmol/L) for the control experiments.²⁶ Both XIP and sXIP were generous gifts from Dr Kenneth Philipson (UCLA, Los Angeles, Calif).

	Results

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The Transient Inward Current Follows Stimulation With a Long Action Potential Waveform
Stimulation using a prolonged action potential waveform reproducibly induced I_ti in 60 of 60 cells (Figure 1A). In some cases, I_ti followed every stimulation using the long action potential waveform, but in other experiments, I_ti occurred in an alternating pattern. Stimulation with a shorter action potential waveform under identical conditions failed to elicit I_ti (n=12) (Figure 1B).

The Transient Inward Current Is Likely Due to the Na⁺/Ca²⁺ Exchanger
I_ti was not observed in any (n=6) of the XIP-treated cells (Figure 2A), but was present in all (n=5) cells treated with sXIP (Figure 2B). In another group of cells, niflumic acid (n=9) failed to prevent I_ti (Figure 2C). These experiments therefore suggest that I_ti may be due to forward-mode Na⁺/Ca²⁺ exchange current. When conventional square wave conditioning steps were used to activate the oscillatory current (Figure 2D and 2E), the reversal potential (E_rev) was between +20 and +40 mV (Figure 2F), which is far different from the calculated E_rev for Cl^– (–45 mV) or monovalent cations (–2 mV) under these experimental conditions. Treatment with niflumic acid only significantly inhibited outward current at +40 and +60 mV compared with control and did not affect inward current or E_rev (Figure 2F). XIP prevented inward current oscillations and shifted E_rev in a positive direction (Figure 2F). The measured E_rev for the oscillatory currents, the lack of effect of niflumic acid, and the suppression of I_ti by XIP are all consistent with the hypothesis that the predominant mechanism underlying I_ti was the Na⁺/Ca²⁺ exchanger.

The [Ca²⁺]_i Transient Is Greater With the Long Than the Short Action Potential Waveform
Integrated fluo 3 fluorescence from paired experiments (Figure 1C) showed that the steady-state [Ca²⁺]_i transient with the long action potential (557±89 Vxms) was significantly greater than that measured during the short action potential (328±28 Vxms) waveform (P=0.04, n=5). These results are consistent with previous reports that show that prolonged action potential duration is associated with increased [Ca²⁺]_i²⁷ and suggest that increased [Ca²⁺]_i is important for I_ti.

The Transient Inward Current Is Associated With Secondary [Ca²⁺]_i Oscillations
Myocytes dialyzed with indo 1 and exhibiting I_ti (n=3) showed a secondary [Ca²⁺]_i oscillation after completion of the long action potential step. Myocytes without I_ti (n=10) did not have secondary [Ca²⁺]_i oscillations. Similar observations were made in fluo 3–loaded cells. These findings provide further evidence that the I_ti measured in this study is dependent on an increase in [Ca²⁺]_i.

The Transient Inward Current Is Dependent on Activator Ca²⁺ From the SR
Addition of BAPTA completely inhibited I_ti after stimulation with the long action potential waveform in 9 of 9 cells studied. Because the release of SR Ca²⁺ stores has been linked to I_ti, I_ti inducibility was tested after exposure to ryanodine (n=6) or thapsigargin (n=6). I_ti was not inducible after exposure to either of these agents. These findings support the hypothesis that I_ti is linked to increased [Ca²⁺]_i and specifically indicates that participation of SR activator Ca²⁺ is necessary for I_ti after stimulation with a prolonged action potential waveform.

The Transient Inward Current Is Dependent on CaM Kinase
To test whether CaM kinase is necessary for I_ti, cells were dialyzed with specific CaM kinase inhibitory peptides. Dialysis with AC3-I (n=11), 273-302 (n=5), or 291-317 (n=4) completely abolished I_ti after stimulation with the prolonged action waveform (Figure 3). Dialysis of the inactive control peptide AC3-C (n=6) had no apparent effect and failed to inhibit I_ti. Like XIP, the CaM kinase inhibitory peptide AC3-I also inhibited oscillatory inward currents in response to test potentials from –80 to +20 mV (Figure 4) but, unlike XIP (Figure 2F), AC3-I did not change the E_rev.

View larger version (33K): [in this window] [in a new window]   Figure 3. Effect of kinase inhibitory peptides on development of Iti. Shown are representative original data tracings of cells stimulated with the long action potential waveform (Figure 1A) after dialysis with inhibitory peptides. Inactive control peptide AC3-C did not prevent Iti (A). The CaM kinase inhibitory peptides AC3-I (B) and 273-302 (C) and the CaM inhibitory peptide 291-317 (D) all prevented Iti. Inhibitory peptides against PKA (E) and PKC (F) failed to prevent Iti.

View larger version (20K): [in this window] [in a new window]   Figure 4. Current-voltage relationship of the oscillatory currents in response to the test voltage commands shown in Figure 2D. The control cells are the same as those shown in Figure 2E. Dialysis with AC3-I inhibited both inward and outward oscillatory currents without changing the apparent Erev. The inactive control peptide AC3-C was without effect.

Both PKA and PKC may also participate in [Ca²⁺]_i homeostasis.^{9 28 29} To test for a possible role of these kinases in I_ti development, cells were dialyzed with suprainhibiting concentrations of protein kinase A inhibitor (PKI 6-22) (n=6) and PKC 19-36 (n=5), and neither of these inhibitory peptides prevented I_ti (Figure 3). These results suggest that I_ti is dependent on CaM and CaM kinase, but not on PKC or PKA, under these experimental conditions.

	Discussion

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Action Potential Prolongation, Arrhythmogenesis, and [Ca²⁺]_i
Action potential prolongation has both antiarrhythmic and proarrhythmic consequences. Since the Cardiac Arrhythmia Suppression Trial³⁰ demonstrated that potent Na⁺ channel blocking agents resulted in excess mortality, considerable effort has been expended to develop safer antiarrhythmic agents. Most of these agents prolong action potential repolarization. With the exception of amiodarone, which has multiple actions, including CaM antagonism,³¹ all of these agents cause significant proarrhythmia. Proarrhythmia from action potential prolongation is thought to result from triggering due to EADs and subsequent functional reentry.²¹

Action potential prolongation results in increased [Ca²⁺]_i (Figure 1C),²⁷ and increased CaM kinase activity^{3 4} ; both of these actions have been linked to EADs.^{4 28} I_ti underlies DADs and is another cause of arrhythmia due to elevated [Ca²⁺]_i and oscillatory release of SR activator Ca²⁺.^{11 32} Because CaM kinase has recently been shown to enhance SR Ca²⁺ release during excitation-contraction coupling,¹ we hypothesized that CaM kinase would facilitate I_ti. The present findings show that a prolonged action potential waveform increases [Ca²⁺]_i under steady-state conditions and results in I_ti. I_ti measured in response to the prolonged action potential waveform has dependence on SR activator Ca²⁺ similar to that of previously measured^{11 32} currents during [Ca²⁺]_i overload under near-physiological conditions.

It is increasingly recognized that prolongation of action potential repolarization is an important cause of increased [Ca²⁺]_i²⁷ and that it is associated with lethal arrhythmias in a variety of settings.^{21 22} Many studies have implicated EADs as an arrhythmogenic trigger when action potential is prolonged. However, most such studies use block of the rapid component of the delayed rectifier (I_Kr) to prolong action potentials, especially at slow rates.²¹ Action potential prolongation that persists at fast heart rates is characteristic of blockade of the slow component of the delayed rectifier (I_Ks), and it is now known that the most common cause of long-QT syndrome is mutations in the KVLQT1 gene, which encodes I_Ks components.²¹ Importantly, preliminary reports have implicated DADs as the arrhythmogenic mechanism when I_Ks is blocked and isoproterenol is added to the experimental preparation.³³ The onset of torsade de pointes occurs with adrenergic stress in >95% of patients with mutations in KVLQT1, according to preliminary reports.³⁴ Our findings suggest a possible mechanism for arrhythmias due to action potential prolongation and point out possible proarrhythmic consequences of using I_Ks blocking agents for antiarrhythmic therapy.

The Identity of the Transient Inward Current
Previous studies have generally concluded that the predominant inward current after a depolarizing step is due to (forward-mode) Na⁺/Ca²⁺ exchanger current in the presence of physiological solutions.^{12 35} However, other candidate currents include I_Ca/Cl and I_CAN.^{12 13 14 15 16 17 18 19} Our findings suggest that I_ti measured in these experiments is due to Na⁺/Ca²⁺ exchanger current for the following reasons. (1) I_ti is inhibited by XIP, but not by the inactive control peptide sXIP (Figure 2). (2) E_rev for the oscillatory current is approximately +30 mV, which is far different from that predicted for I_Ca/Cl (–45 mV) or I_CAN (–2 mV) but consistent with previously reported values under similar experimental conditions, in which both the Na⁺/Ca²⁺ exchanger current and I_Ca/Cl were present.^{17 36} (3) I_ti is not inhibited by niflumic acid (Figure 2C). Niflumic acid did reduce outward currents at positive potentials (Figure 2F), which is consistent with the described outward rectification of I_Ca/Cl.¹⁹ These results suggest that I_ti is due to the Na⁺/Ca²⁺ exchanger and that I_Ca/Cl is present but significant only at relatively depolarized cell membrane potentials under these conditions. One recent report in ferret ventricular myocytes found that the time course for activation of the Na⁺/Ca²⁺ exchanger current was slower than for I_Ca/Cl, making E_rev measurement problematic.³⁷ Limitations to our conclusion that I_ti is due to the Na⁺/Ca²⁺ exchanger in these experiments are that (1) no antagonists are available to exclude participation of I_CAN, and (2) XIP is a CaM antagonist over the range of concentrations useful for inhibition of the Na⁺/Ca²⁺ exchanger.³⁸ Thus, inhibition of I_ti by XIP could occur partially via a CaM kinase–dependent mechanism analogous to the inhibition with the CaM inhibitory peptide 291-317 (Figure 3D). It is interesting to note that XIP shifted the apparent E_rev for the oscillatory current (Figure 2F), whereas the specific CaM kinase inhibitory peptide AC3-I did not (Figure 4), which suggests that XIP effects are not solely due to CaM kinase inhibition and therefore likely reflect inhibition of the Na⁺/Ca²⁺ exchanger.

CaM Kinase Inhibition
The specific inhibitory peptides used in these experiments offer a significant advantage over other organic inhibitors, because the latter are also I_Ca-L antagonists.^{1 4 5} Two different peptides modeled after separate regions of the CaM kinase molecule were effective at suppressing I_ti (Figure 3B, 3C, and 4). A less specific inhibitory peptide with antagonist actions against CaM (and presumably a diverse array of CaM-dependent processes, including CaM kinase) was also effective at preventing I_ti (Figure 3D). Thus, inhibitory peptides acting at 3 different sites along the CaM kinase activation pathway were all effective in preventing I_ti. In contrast, inhibitory peptides against PKA or PKC did not prevent I_ti. Although these studies do show that CaM kinase activity appears necessary for I_ti, the results do not precisely define or quantify the site(s) of action of CaM kinase inhibition for I_ti suppression. One possibility is that enhanced [Ca²⁺]_i activates CaM kinase, which, in turn, augments SR Ca²⁺ uptake and release to secondarily activate I_ti caused by forward-mode Na⁺/Ca²⁺ exchange. These results strongly support the hypothesis that CaM kinase facilitates I_ti due to the Na⁺/Ca²⁺ exchanger during increased [Ca²⁺]_i. CaM kinase may thus be a useful antiarrhythmic drug target to prevent arrhythmias related to [Ca²⁺]_i overload.

	Acknowledgments

 
This work was supported by NIH Grants HL03727 (to M.E.A.) and HL46681 and HL49989 (to D.M.R.) and a Cardiac Arrhythmia Research and Education Foundation, Inc, award (to M.E.A.). D.M.R. holds the William Stokes chair in Experimental Therapeutics, a gift of the Dai-ichi Corp.

Received October 1, 1998; accepted February 1, 1999.

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