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Am J Physiol Heart Circ Physiol 276: H2168-H2178, 1999;
0363-6135/99 $5.00
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Vol. 276, Issue 6, H2168-H2178, June 1999

CaM kinase augments cardiac L-type Ca2+ current: a cellular mechanism for long Q-T arrhythmias

Yuejin Wu1, Leigh B. MacMillan2, R. Blair McNeill2, Roger J. Colbran2, and Mark E. Anderson1,3

Departments of 1 Medicine, 2 Molecular Physiology and Biophysics, and 3 Pharmacology, Vanderbilt University, Nashville, Tennessee 37232-6300


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Early afterdepolarizations (EAD) caused by L-type Ca2+ current (ICa,L) are thought to initiate long Q-T arrhythmias, but the role of intracellular Ca2+ in these arrhythmias is controversial. Rabbit ventricular myocytes were stimulated with a prolonged EAD-containing action potential-clamp waveform to investigate the role of Ca2+/calmodulin-dependent protein kinase II (CaM kinase) in ICa,L during repolarization. ICa,L was initially augmented, and augmentation was dependent on Ca2+ from the sarcoplasmic reticulum because the augmentation was prevented by ryanodine or thapsigargin. ICa,L augmentation was also dependent on CaM kinase, because it was prevented by dialysis with the inhibitor peptide AC3-I and reconstituted by exogenous constitutively active CaM kinase when Ba2+ was substituted for bath Ca2+. Ultrastructural studies confirmed that endogenous CaM kinase, L-type Ca2+ channels, and ryanodine receptors colocalized near T tubules. EAD induction was significantly reduced in current-clamped cells dialyzed with AC3-I (4/15) compared with cells dialyzed with an inactive control peptide (11/15, P = 0.013). These findings support the hypothesis that EADs are facilitated by CaM kinase.

arrhythmia; calcium channels; action potential; long Q-T syndrome; sarcoplasmic reticulum


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXCESSIVE CARDIAC ACTION POTENTIAL prolongation, demonstrated clinically by Q-T interval prolongation, is associated with an increased intracellular Ca2+ concentration ([Ca2+]i) transient (3) and ventricular arrhythmias (26). Early afterdepolarizations (EADs) are oscillations in cell membrane voltage during action potential repolarization, caused by net inward current, and are generally thought to initiate arrhythmias caused by Q-T interval prolongation, including torsade de pointes (26). Slowly inactivating L-type Ca2+ current (ICa,L) and L-type Ca2+ window current have been implicated as sources of inward current for EADs (21). Because of the potential involvement of interdependent currents, it has not been a straightforward process to determine the role of [Ca2+]i on ICa,L in EADs.

We have produced evidence that the activation of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase) may be a crucial link between increased action potential duration (APD) and EAD-related arrhythmias. CaM kinase activity is known to be augmented by increased [Ca2+]i, and we have found that CaM kinase augmented ICa,L (1), that CaM kinase activity increased when APD was prolonged (2), and that in isolated hearts EADs were blocked by CaM kinase inhibition (2). Still, the role of [Ca2+]i in ICa,L-related EADs remains controversial (22). In the present study we have used an EAD-containing action potential waveform to directly measure the ICa,L associated with action potential repolarization and EADs. This procedure allows for the isolation and direct measurement of arrhythmogenic ICa,L over a physiologically relevant range of cell membrane potentials. We present evidence that CaM kinase mediates an increase in ICa,L by a mechanism dependent on sarcoplasmic reticulum (SR) Ca2+ stores and that CaM kinase inhibition decreases EADs. These findings lend further support to the hypothesis that CaM kinase is a proarrhythmic signaling molecule during action potential prolongation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myocyte isolation. Isolation of rabbit ventricular myocytes was performed as previously described (1). New Zealand White rabbits of either gender (2-3 kg) were killed with a pentobarbital sodium overdose (50 mg/kg iv) after heparin infusion (150 U/kg iv). The collagenase-containing solution was prepared in nominally Ca2+-free saline containing 60 U/ml collagenase (type I, Worthington Biochemicals, Freehold, NJ) and 0.1 U/ml protease (type XIV, Sigma, St. Louis, MO). Only rod-shaped quiescent myocytes from the left ventricle with clear striations were studied.

Solutions. Action potential recording was performed in the following bath solution (in mmol/l): 140.0 NaCl, 10.0 glucose, 5.0 HEPES, 5.4 KCl, 2.5 CaCl2, and 1.0 MgCl2; pH was adjusted to 7.4 with 10 N NaOH. Action potential prolongation and EAD induction followed reduction of the bath solution KCl to 3.0 mmol/l and the use of a long (10 s) interstimulus interval. The intracellular pipette solution for action potential recording contained (in mmol/l) 120.0 K-aspartate, 10.0 HEPES, 10.0 EGTA, 5.0 Na2ATP, 4.0 MgCl2, and 3.0 CaCl2; pH was adjusted to 7.2 with 1 N KOH. Unless otherwise noted, all chemicals were from Sigma.

L-type Ca2+ current recordings were performed in 0 Na+ bath solution containing (in mmol/l) 137.0 choline-Cl or N-methyl-D-glucamine (NMDG), 20.0 or 25.0 CsCl, 10.0 HEPES, 10.0 glucose, 5.4 KCl, 1.8 CaCl2, and 0.5 MgCl2; pH was adjusted to 7.4 with 1.0 N CsOH. In some experiments Ba2+ (1.8 mmol/l) was substituted for Ca2+ after the attainment of whole cell mode configuration (see below). Nifedipine (10 µmol/l) or Cd2+ (2.0 mmol/l) was added to the bath solution at the end of all experiments, and niflumic acid (10 µmol/l) was included for most experiments to inhibit a Ca2+-sensitive chloride current (31, 36). Thapsigargin (100 nmol/l; Calbiochem, La Jolla, CA) (32) or ryanodine (1.0 or 10.0 µmol/l) (11) were included in some experiments to inhibit participation of SR stores in [Ca2+]i homeostasis. Both ryanodine concentrations had identical effects on ICa,L, so the results of these experiments were pooled and reported together. The intracellular pipette solution contained (in mmol/l): 120.0 CsCl, 10.0 EGTA, 10.0 HEPES, 10.0 tetraethylammonium chloride (TEA), 5.0 phosphocreatine, 3.0 CaCl2, 1.0 MgATP, and 1.0 NaGTP; pH was adjusted to 7.2 with 1.0 N CsOH. The calculated resting free [Ca2+]i with this solution was ~100 nmol/l (1). The fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; 20.0 mmol/l) was substituted for EGTA to enhance [Ca2+]i buffering in some experiments. Both EGTA and CaCl2 were omitted during [Ca2+]i-monitoring studies (see below). Isoproterenol was mixed fresh and used at a final bath concentration of 40 nmol/l for protein kinase A (PKA) inhibitor peptide control experiments. The CaM kinase inhibitor KN-93 (0.5-1.0 µmol/l) was added to the bath for some experiments (2).

Current clamp. Cells were stimulated at 0.1 Hz in whole cell configuration in current-clamp mode with 0.1- to 1.0-nA pulses of depolarizing current (1.25× threshold) for 2-3 ms at room temperature (20-23°C) for 5-100 beats. Bath K+ was reduced to 3.0 mmol/l to favor action potential prolongation and EADs. APDs were quantified as the time from phase 0 at 50% (APD50) and 90% (APD90) repolarization. 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; Axon Instruments, Foster City, CA). A long action potential waveform with an EAD was digitized and stored for application as a voltage command using pCLAMP 6.03 (Axon Instruments) (Fig. 1A).


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  		Fig. 1.   L-type Ca2+ current (ICa,L) evoked in response to voltage clamp with a prolonged action potential waveform. A: action potential waveform used as a voltage-clamp command in studies. Waveform was generated in current-clamp mode where repolarization was prolonged after reduction of extracellular K+ and slow (0.1 Hz) stimulation. An early afterdepolarization (EAD) is seen as an upward oscillation in the repolarization plateau (phase 2). Vm, membrane potential. B: L-type Ca2+ current evoked in response to action potential command waveform in whole cell voltage clamp. Two distinct components of ICa,L are present at repolarization-associated cell membrane potentials: ICa,L1 and ICa,L2; ICa,L2 is in response to the EAD waveform. Dashed vertical lines indicate bounds of ICa,L1 and ICa,L2. Both ICa,L components initially increase with pacing (0.5 Hz), and both are inhibited by nifedipine (10 µmol/l). C: nifedipine-sensitive difference currents from B. D: time course of biphasic responses (i.e., initial augmentation followed by inhibition) of ICa,L1 and ICa,L2 to pacing at 0.5 Hz (n = 5). L-type Ca2+ current (i.e., nifedipine-sensitive difference current) is integrated and expressed as total charge movement corrected for cell size.

Voltage clamp. All studies were performed at room temperature with patch-clamp methodology in the whole cell configuration (16) using an Axopatch 200B amplifier (Axon Instruments). Micropipette electrode resistance was 1.0-2.5 MOmega when filled with the intracellular solution. Membrane currents were low-pass filtered at 2 kHz and sampled at 18.5 kHz as described in Current clamp. The digitized signals were stored in a microcomputer for later analysis with pCLAMP 6.03 and SigmaPlot (Jandel Scientific, Chicago, IL). After a period (>= 5 min) of quiescent dialysis and stabilization, cells were repetitively depolarized at 0.1 or 0.5 Hz with the action potential command waveform. Cell membrane capacitance was measured using the integral of the current transient after a 10-mV hyperpolarizing step from a holding potential of -80 mV (166 ± 2.3 pF).

Studies using action potential clamp are complicated by constantly changing cell membrane potential, leakage, and capacitive currents (31). In these experiments, ICa,L was completely inhibited by the addition of Cd2+ (2 mmol/l) or nifedipine (10 µmol/l) to the bath solution (Fig. 1B). The residual capacitive and leakage current was subtracted from all records using pCLAMP 6.03 before analysis so that only nifedipine (or Cd2+)-sensitive difference currents were analyzed. A small current [6.2 ± 0.2% of the first component of ICa,L (ICa,L1)], presumably the Ca2+-activated Cl- current, was inhibited by the addition of niflumic acid (10 µmol/l) in most experiments (31, 36). The component of ICa,L elicited in response to the rapid depolarization phase of the action potential clamp waveform was not included in the analysis (Fig. 1B). Two distinct components of ICa,L were present during repolarization in response to the action potential waveform used in this study (Fig. 1B). The larger, first component (ICa,L1) was present during most of phase 2 repolarization. Plateau repolarization and ICa,L1 were interrupted by a depolarizing oscillation (EAD) that marked the onset of the second ICa,L component (ICa,L2; Fig. 1, A-C). Inflection points on the ICa,L waveform were used to define the onset of ICa,L1 and ICa,L2 (Fig. 1, B and C). Both ICa,L1 and ICa,L2 were then integrated using pCLAMP 6.03, and the results were expressed as total charge movement.

Inhibitor peptides. CaM kinase, PKA, and protein kinase C (PKC) were inhibited in separate experiments by dialyzing the quiescent cell for >= 5 min with kinase inhibitor peptides before the experimental protocol was initiated. CaM kinase was inhibited with AC3-I (KKALHRQEAVDCL; 20 µmol/l) (5). The amino acid sequence HRQEAVDC corresponds to that surrounding the autophosphorylation site (Thr-286/287) on CaM kinase, except that T is modified to A (Ala) to prevent phosphorylation. Thus AC3-I is a modified CaM kinase substrate that inhibits substrate phosphorylation (in vitro IC50 ~3 µmol/l) by activated CaM kinase. Other serine/threonine kinases are not significantly inhibited by AC3-I at the concentration used in these experiments (5). The peptide AC3-C (KKALHAQERVDCL) has no inhibitory activity (5) and was included in the pipette solution (20 µmol/l) in separate control experiments. AC3-I and AC3-C were prepared using a solid-phase peptide synthesizer (Applied Biosystems) and purified by reversed-phase high-performance liquid chromatography. The sequences were confirmed by automated sequencing. Both AC3-I and AC3-C were generous gifts from Dr. Howard Schulman, Stanford University, Stanford, CA.

The competitive PKA inhibitory (PKI) peptide (IC50 = 0.2 µmol/l) corresponds to positions 6-22 (TYADFIASGRTGRRNAI) of PKI and was included in the pipette solution at 1.0 µmol/l. PKI was effective at suppressing isoproterenol-induced ICa,L augmentation under these experimental conditions (n = 4). The competitive PKC inhibitor peptide (IC50 = 0.3 µmol/l) corresponds to the autoinhibitory domain positions 19-36 (RFARKGALRQKNVHEVKN) of PKC and was included in the pipette solution at 20 µmol/l. Both PKA and PKC inhibitor peptides were obtained commercially (GIBCO BRL, Gaithersburg, MD).

Constitutively active CaM kinase. A recombinant monomeric truncation mutant of the mouse CaM kinase II alpha -isoform (amino acid residues 1-380) was expressed using baculovirus and then purified using CaM agarose affinity chromatography. Its properties were essentially the same as a 1-316 truncated mutant described previously (7) and will be described in detail elsewhere. The CaM kinase II(1-380) [stored in 50 mmol/l HEPES, pH 7.5, 1 mmol/l EDTA, 1 mmol/l DTT, 50% (vol/vol) glycerol, and 10% (vol/vol) ethylene glycol] was activated by autophosphorylation in a 100-µl reaction containing 50 mmol/l HEPES, pH 7.5, 2 mmol/l magnesium acetate, 1.5 mmol/l CaCl2, 18 µmol/l CaM, 2 mmol/l DTT, and 100 µmol/l adenosine 5'-O-(3-thiotriphosphate). The reaction was initiated with the addition of the CaM kinase II(1-380) (9 µmol/l final subunit concentration) followed by incubation at 30°C for 10 min. The reaction was stopped with the addition of EDTA (10 mmol/l). Ca2+/CaM-dependent autophosphorylation of the CaM kinase II produces an active species that can phosphorylate substrates in the absence of Ca2+/CaM. This Ca2+-independent or autonomous activity of the autophosphorylated CaM kinase II(1-380) was evaluated against the peptide substrates syntide-2 and autocamtide and was typically 35-50% of total activity in the presence of Ca2+/CaM. Autophosphorylated kinase was diluted 10- to 100-fold in the ICa,L pipette solution (0.9-0.09 µmol/l final) used in voltage-clamp studies and its activity confirmed in vitro. In vitro enzyme activity was maintained (>75% of initial activity over 6 h) throughout the voltage-clamp experiments. This dilution was chosen to approximate the physiological CaM kinase activity in heart (~1-2 µmol/l) derived from percent yield calculations during purification (15, 19). CaM kinase II (0.9-0.09 µmol/l final) was heated to 70°C for 15 min for control experiments, and a lack of kinase activity was confirmed as described above.

Fluo 3 fluorescence. Intracellular Ca2+ was monitored during some experiments by the inclusion of the pentapotassium salt of the fluorescent Ca2+ indicator fluo 3 (Molecular Probes, Eugene, OR) in the pipette solution (100 µmol/l) as previously described, with minor modifications (1). Cells were dialyzed for an average of 5 min in whole cell mode to achieve an equilibrium between the pipette and intracellular solutions, and voltage signals were low-pass filtered at 50 Hz before analysis. Fluo 3 [Ca2+]i transients were integrated after resting [Ca2+]i levels were normalized to 0 V to cancel any potential contribution of increasing intracellular fluo 3 concentration to the fluorescent signal during the course of an experiment (pCLAMP 6.03). Analysis of peak transients without this correction [i.e., fluorescence at time indicated (F)/baseline fluorescence (Fo)] yielded a qualitatively similar pattern of increase with stimulation.

Immunofluorescence staining. Freshly isolated rabbit ventricular myocytes were seeded onto glass coverslips (2.25 × 104 cells/coverslip) and sedimented by centrifugation (1,800 g, 30 min, 15°C). Myocytes were washed twice with relaxation buffer [0.1 mol/l KCl, 5 mmol/l EGTA, 5 mmol/l MgCl2, and 0.25 mmol/l DTT in phosphate-buffered saline (PBS), pH 6.8] and were then fixed in precooled (-20°C) methanol-acetone (1:1) for 10 min at 4°C. Fixed myocytes were washed once with PBS and incubated in labeling buffer (1% bovine serum albumin, 2% normal donkey serum, and 0.1% Triton X-100 in PBS) for at least 2 h at room temperature to block nonspecific binding. Primary antibodies were diluted in labeling buffer and incubated with myocytes overnight at 4°C. Primary antibodies included the previously described goat anti-CaM kinase (23) and monoclonal anti-alpha -actinin, which stains Z lines and thus indicates T tubules (14). After at least five washes with PBS containing 0.1% Triton X-100, secondary antibodies (Cy3-conjugated donkey anti-goat and Cy2-conjugated donkey anti-mouse diluted in wash buffer) were incubated with myocytes for 2 h at room temperature. After at least five washes, coverslips were mounted on slides using Aqua-PolyMount (Polysciences, Warington, PA). Fluorescence was visualized on a confocal microscope (Carl Zeiss, Oberkochen, Germany). Negative control experiments were performed using secondary antibodies alone, and no specific staining was observed (data not shown).

Statistics. Data were analyzed using a paired Student's t-test, a Fisher's exact test (for EAD inducibility rates), or ANOVA as appropriate. The null hypothesis was rejected at the 0.05 level.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Both ICa,L1 and ICa,L2 exhibit similar biphasic responses to pacing. The prolonged action potential waveform containing an EAD used for a voltage-clamp command for part of this study is shown in Fig. 1A. The nifedipine (10 µmol/l)- or Cd2+ (2 mmol/l)-sensitive currents were referred to as ICa,L. The initial current component was associated with rapid depolarization during the action potential upstroke. However, two much larger components of ICa,L were clearly distinguished (Fig. 1B) during the repolarization phase of the action potential waveform. The first component (ICa,L1) was larger than the second, EAD-associated component (ICa,L2), but both components had a similar biphasic response to pacing that consisted of initial augmentation followed by inhibition to below the initial value (Fig. 1D). This biphasic ICa,L response was similar during pacing at 0.1 and 0.5 Hz.

Intracellular Ca2+ continuously increases during pacing. In contrast to the biphasic response of ICa,L1 and ICa,L2 to repetitive stimulation, bulk cytoplasmic [Ca2+]i continuously increased. Myocytes were dialyzed with the fluorescent Ca2+ indicator fluo 3 during voltage-clamp studies to measure the response of the [Ca2+]i transient during pacing (Fig. 2A, n = 6). Under these reduced [Ca2+]i buffering conditions, ICa,L1 and ICa,L2 increased and decreased with stimulation similar to the results shown in Fig. 1D. When the area under the fluorescence transient was integrated and plotted against the beat number, it was apparent that [Ca2+]i transients became progressively larger, but a significant increase was present by the second stimulated beat (Fig. 2B).


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  		Fig. 2.   Intracellular Ca2+ concentration ([Ca2+]i) increases during pacing. A: fluo 3 fluorescence measurement of an [Ca2+]i transient. Transients from 1st and 20th beats are superimposed for comparison. Photomultiplier output is in volts. B: time course of integrated fluo 3 fluorescence [Ca2+]i transient during pacing at 0.5 Hz (n = 5). * Significant increase over 1st beat in A.

The biphasic responses of ICa,L1 and ICa,L2 to pacing are dependent on [Ca2+]i. A reproducible and marked increase in both ICa,L1 and ICa,L2 was present during the initial beats in myocytes paced at 0.1 Hz (data not shown) and 0.5 Hz under control conditions followed by a secondary inhibitory phase (Fig. 1). The experiments shown in Fig. 3 suggest that these biphasic changes in ICa,L1 and ICa,L2 were dependent on [Ca2+]i because 1) initial augmentation was prevented and 2) the secondary inhibition markedly slowed in a separate group of myocytes dialyzed with the Ca2+ chelator BAPTA (Fig. 3, A and D). In contrast, only the initial augmentation phase of ICa,L1 and ICa,L2 to pacing was abolished in cells exposed to the SR Ca2+-release channel antagonist ryanodine (Fig. 3B) or the SR Ca2+-ATPase antagonist thapsigargin (Fig. 3C). The increased magnitude of ICa,L at the first stimulated beat is consistent with depletion of SR Ca2+ stores (31). These findings suggest that both augmentation and inhibition of ICa,L1 and ICa,L2 are dependent on [Ca2+]i but that augmentation is specifically linked to the release of Ca2+ from the SR.


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  		Fig. 3.   Effect of altered [Ca2+]i homeostasis on biphasic responses of ICa,L1 (filled circles) and ICa,L2 (open circles) to pacing. Small filled circles in A-C are data points scaled to peak ICa,L1 and ICa,L2 from control cells shown in Fig. 1D for comparison, and data are expressed as in Fig. 1D. A: enhanced [Ca2+]i buffering by dialysis with 20 mmol/l 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (n = 6) abolishes initial augmentation and markedly slows the rate of secondary inhibition for ICa,L1 and ICa,L2 compared with control conditions. B and C: inhibition of sarcoplasmic reticulum Ca2+ release by ryanodine (10 µmol/l, n = 12; B) or Ca2+ uptake by thapsigargin (100 nmol/l, n = 10; C) abolishes initial augmentation responses of ICa,L1 and ICa,L2 to pacing without altering the pattern of secondary inhibition. D: summary of initial augmentation response of ICa,L1 and ICa,L2 to pacing (0.5 Hz) shown in A-C. Complete inhibition of ICa,L1 and ICa,L2 augmentation by BAPTA, ryanodine, and thapsigargin demonstrates that the process(es) underlying ICa,L augmentation is dependent on SR activator Ca2+. Both ICa,L components are integrated and expressed as the ratio of maximum charge movement (Qmax) to charge movement at the 1st beat (Q1). * Pacing significantly augmented ICa,L1 (P = 0.01) and ICa,L2 (P = 0.02) only in control group (n = 6).

CaM kinase is necessary for augmentation of ICa,L1 and ICa,L2 by [Ca2+]i. To test the hypothesis that CaM kinase mediates the increase in ICa,L during action potential repolarization in response to increased [Ca2+]i, a control group of myocytes was dialyzed with the inactive peptide AC3-C (Fig. 4A), whereas other myocytes were dialyzed with the specific CaM kinase inhibitor peptide AC3-I (Fig. 4B) (5). The striking biphasic pattern of early ICa,L augmentation and subsequent inhibition was maintained in the presence of the inactive control peptide (Fig. 4A). However, the CaM kinase inhibitor peptide AC3-I completely prevented the increase in both ICa,L1 and ICa,L2 without any major effect on the secondary pattern of inhibition (Fig. 4B). ICa,L augmentation was also prevented in cells (n = 3) treated with the nonpeptide cell membrane permeant CaM kinase inhibitory agent KN-93 (data not shown).


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  		Fig. 4.   Effect of Ca2+/calmodulin-dependent protein kinase II (CaM kinase), protein kinase C (PKC), and protein kinase A (PKA) inhibition on responses of ICa,L1 and ICa,L2 to pacing. Small circles in A-D are scaled data points from control cells (no peptide in dialysate) shown in Fig. 1D for comparison, and data are expressed as in Fig. 1D. A: biphasic responses of ICa,L1 and ICa,L2 to pacing are not affected by dialysis with inactive control peptide AC3-C (20 µmol/l, n = 7). B: dialysis with CaM kinase inhibitor peptide AC3-I (20 µmol/l, n = 7) completely prevented augmentation of ICa,L1 and ICa,L2 but was without effect on secondary inhibitory phase during pacing. C and D: dialysis with PKC inhibitor peptide PKC 19-36 (20 µmol/l, n = 17) or PKA inhibitor peptide PKA 6-22 (1 µmol/l, n = 18) did not prevent biphasic response of ICa,L1 or ICa,L2 to pacing. E: summary of ICa,L1 and ICa,L2 augmentation responses to pacing in cells dialyzed with peptides from A-D. Data are expressed as in Fig. 3D. * All groups showed a significant increase in both ICa,L1 and ICa,L2 with pacing except for cells dialyzed with CaM kinase inhibitor peptide AC3-I: AC3-C, P = 0.001 for ICa,L1 and P = 0.01 for ICa,L2; PKC 19-36, P < 0.001 for ICa,L1 and P < 0.001 for ICa,L2; and PKA 6-22, P < 0.001 for ICa,L1 and P < 0.001 for ICa,L2.

The serine/threonine kinases PKC and PKA also have the potential to augment ICa,L (20) and are stimulated by Ca2+-dependent mechanisms in some tissues (4, 30). Therefore, separate experiments were performed in myocytes dialyzed with peptide inhibitors against PKC (n = 13) and PKA (n = 15). Unlike the effect of CaM kinase inhibition, the general biphasic responses of both ICa,L1 and ICa,L2 to pacing persisted in PKC (Fig. 4C) and PKA (Fig. 4D) inhibitor peptide-treated cells. Although both PKC and PKA inhibitor peptides did reduce the maximum percent increase in ICa,L components with pacing, these increases persisted and remained significant (Fig. 4E). Slowing of the secondary inhibitory response to pacing was also seen with both the PKC and PKA inhibitor peptides. The magnitude of ICa,L at the first beat was not different in cells treated with control or inhibitor peptides. These findings demonstrate that CaM kinase, but not PKA or PKC, transduces most or all of the increase in ICa,L1 and ICa,L2 during prolonged repolarization-associated cell membrane potential command steps under these experimental conditions.

CaM kinase is partially localized to T tubules in rabbit ventricular myocytes. An emerging theme in ion-channel regulation is that ion channels are intimately associated with the protein kinases involved in their regulation (24). Our data suggest that L-type Ca2+ channel activity is augmented by a mechanism involving the release of SR Ca2+ and the activation of CaM kinase. We therefore investigated whether CaM kinase was localized within ventricular myocytes at a site where it could be expected to mediate the observed augmentation of ICa,L. As shown in Fig. 5, immunofluorescence staining demonstrated partial colocalization of CaM kinase with alpha -actinin; CaM kinase is also localized along longitudinal structures, presumably the SR (Fig. 5A), and is diffusely cytosolic. We confirmed previous observations (8) that L-type Ca2+ channels and ryanodine receptors are also colocalized on the Z lines (not shown). These studies indicate that CaM kinase is partially colocalized with L-type Ca2+ channels and ryanodine receptors along the T tubules in these myocytes. This ultrastructural relationship is anticipated to favor augmentation of ICa,L by a mechanism involving SR activator Ca2+ and CaM kinase.


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  		Fig. 5.   Subcellular localization of CaM kinase in rabbit ventricular myocytes. Myocytes were labeled with antibodies specific for CaM kinase IIa (CaMKII; A) and alpha -actinin (B). Images were visualized using Cy3 (red)- and Cy2 (green)-conjugated secondary antibodies and a laser scanning confocal microscope (Carl Zeiss). C: merged image produced by superimposing images in A and B. CaM kinase and alpha -actinin partially colocalize in a strongly striated pattern, indicating concentration at the Z band in area of T tubules.

Constitutively active CaM kinase reconstitutes augmentation of ICa,L1 and ICa,L2 in response to pacing with Ba2+ as charge carrier. The above-described experiments using CaM kinase inhibitor peptides suggested that CaM kinase was required to augment ICa,L by pacing. One possible explanation for this result is that SR Ca2+ activates CaM kinase, which in turn augments ICa,L. To test this hypothesis, Ba2+ was used to measure ICa,L (IBa,L) responses to pacing, independent of endogenous CaM kinase activation, because Ba2+ is a permeant ion in L-type Ca2+ channels (10) but does not activate CaM kinase (9). Ba2+ currents differed in appearance from Ca2+ currents because of their slower inactivation rates (10) but remained easily separable into two components (IBa,L1 and IBa,L2) (Fig. 6A). The IBa,L currents were inhibited by nifedipine (10 µmol/l), and the data refer to ICa,L antagonist-sensitive difference currents (Fig. 6A). As predicted, based on the inability of Ba2+ to substitute for Ca2+ for CaM kinase activation, neither IBa,L1 or IBa,L2 augmented with pacing (Fig. 6, C and D).


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  		Fig. 6.   Effect of constitutively active CaM kinase with Ba2+ as charge carrier on ICa,L1 and ICa,L2 during pacing. A: digitized action potential waveform shown in Fig. 1A was used as a voltage command as in all previous experiments except that extracellular Ba2+ was substituted for Ca2+ and cell was dialyzed with monomeric constitutively active (Ca2+ independent) CaM kinase for 5-10 min before pacing was initiated (0.5 Hz). As in Fig. 1B, 2 components of repolarization-associated Ba2+ current (IBa,L) were present: IBa,L1 and IBa,L2. Addition of constitutively active CaM kinase resulted in IBa,L1 and IBa,L2 augmentation with pacing. Currents through L-type Ca2+ channels were blocked by nifedipine (10 µmol/l). Vertical dashed lines show boundaries for IBa,L1 and IBa,L2. B: nifedipine-sensitive difference currents from A. C: time course of biphasic responses of IBa,L1 (filled symbols) and IBa,L2 (open symbols) to pacing at 0.5 Hz in cells dialyzed with constitutively active CaM kinase (0.9 µmol/l, n = 11; circles) and in cells dialyzed with heat-inactivated CaM kinase (0.9 µmol/l, n = 22; squares) as a control. IBa,L is integrated and expressed as total charge movement corrected for cell size (QBa,L). D: summary of initial augmentation responses of IBa,L1 and IBa,L2 to pacing. Both IBa,L components are expressed as Qmax/Q1. Control, cells (n = 5) not treated with any peptide; Buffer, cells (n = 6) dialyzed with pipette containing CaM kinase buffer but no peptide; AC3-I, cells (n = 6) dialyzed with constitutively active CaM kinase (0.9 µmol/l) and CaM kinase inhibitor peptide AC3-I (20 µmol/l); Heated, cells (n = 22) dialyzed with heat-inactivated constitutively active CaM kinase (0.9 µmol/l); 0.09 µM (n = 4) and 0.9 µM (n = 11), cells dialyzed with 0.09 and 0.9 µmol/l, respectively, of constitutively active CaM kinase. * Significant initial augmentation in IBa,L1 (P = 0.009) or IBa,L2 (P = 0.006) with pacing.

Dialysis with constitutively active CaM kinase would reconstitute the augmentation of IBa,L by pacing if CaM kinase activation alone were sufficient for this response. As shown in Fig. 6, dialysis with constitutively active CaM kinase (0.9 µmol/l) did, in fact, reconstitute the augmentation of both IBa,L1 and IBa,L2 to pacing (Fig. 6C). The action of the constitutively active CaM kinase on IBa,L is likely specifically due to kinase activity because IBa,L augmentation was not observed in cells dialyzed with a lower concentration (0.09 µmol/l) of constitutively active CaM kinase, with heat-inactivated CaM kinase, or when the inhibitor peptide AC3-I (20 µmol/l) was coadministered with the constitutively active CaM kinase (0.9 µmol/l) (Fig. 6C). Substitution of Ba2+ for Ca2+ did not prevent the secondary pattern of current decrease with pacing. Reconstitution of IBa,L augmentation by exogenous constitutively active CaM kinase supports the hypothesis that CaM kinase activation is sufficient for ICa,L augmentation with pacing. These findings are complementary to the experiments with CaM kinase inhibitor peptides and lend further support to the hypothesis that CaM kinase augments repolarization associated ICa,L.

CaM kinase inhibition reduces EAD inducibility. To directly test the hypothesized relationship between CaM kinase activity and EADs, ventricular myocytes were bathed in a reduced K+ solution (3.0 mmol/l) to facilitate action potential lengthening and EAD induction in current-clamp mode (Fig. 7). EAD induction was significantly more frequent in cells dialyzed with the inactive control peptide AC3-C (11/15) than in cells dialyzed with the CaM kinase inhibitor peptide AC3-I (4/15). EADs present in control cells were also more likely to consist of multiple oscillations (6/15) compared with cells treated with the inhibitor peptide (0/15). The emergence of all EADs was early in the stimulation sequence (10/15 in the first 5 beats, and the rest within 28 beats). Suppression of EADs by a CaM kinase inhibitor peptide strongly supports the hypothesis that CaM kinase is a proarrhythmic signaling molecule.


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  		Fig. 7.   Sequential action potential tracings from cells paced in current-clamp mode (0.1 Hz) and bathed in low extracellular K+ (3.0 mmol/l) to favor EAD induction. A: a cell dialyzed with inactive peptide AC3-C developed a single EAD (2nd beat, 1st tracing), followed by multiple EADs (3rd and subsequent beats, 1st tracing), prolonged depolarizations (1st beat, 2nd tracing), and a rise in resting potential and pacing threshold. B: another cell dialyzed with AC3-C initially developed a single EAD (1st beat, 1st tracing), followed by multiple EADs (3rd beat, 3rd tracing). C: a cell dialyzed with CaM kinase inhibitor peptide AC3-I failed to develop EADs. Data for EAD inducibility are summarized in text.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

L-type Ca2+ current measured during voltage clamp with the action potential command waveform. An action potential waveform with a prolonged plateau (phase 2) and an EAD was chosen for the voltage-clamp command under conditions shown to isolate ICa,L, because ICa,L is thought to be arrhythmogenic during action potential prolongation and phase 2 EADs (2, 21, 26). Whereas it is known that ICa,L current is augmented by isoproterenol and the dihydropyridine receptor agonist BAY K 8644 in Purkinje fibers at cell membrane potentials associated with action potential repolarization (17), augmentation of cardiac ICa,L by increased [Ca2+]i at these cell membrane potentials, during the time course of a prolonged action potential, has not been reported. The use of the EAD-containing prolonged action potential waveform provides important new evidence that [Ca2+]i-dependent ICa,L augmentation can occur at cell membrane potentials relevant to EADs and repolarization. The augmentation of ICa,L by increased [Ca2+]i that we have observed is especially important in the setting of prolonged action potential repolarization because action potential prolongation itself results in increased [Ca2+]i (3). Previous work by us (1) and others (33, 35) has demonstrated that CaM kinase augments ICa,L in response to increased [Ca2+]i in ventricular myocytes. Thus we hypothesized that CaM kinase functions as a proarrhythmic signaling molecule during action potential prolongation.

Increased [Ca2+]i is necessary for the biphasic responses of ICa,L1 and ICa,L2 to pacing. The composition of the pipette and bath solutions favored increased [Ca2+]i because the near absence of Na+ in the bath and pipette solutions reduced the ability of the Na+/Ca2+ exchanger to extrude Ca2+ from the cell. Indeed, measurements with fluo 3 confirmed that [Ca2+]i rose continuously during pacing (Fig. 2B) and that the decline of the [Ca2+]i back to baseline was delayed (Fig. 2A) under these experimental conditions. In addition, enhanced cytoplasmic Ca2+ buffering with BAPTA blocked the initial augmentation and slowed the secondary inhibition of ICa,L1 and ICa,L2 (Fig. 3, A and D), indicating a requirement for increased [Ca2+]i for both these responses to pacing. It is interesting to note that other workers (18) have found partial persistence of [Ca2+]i-dependent activation of ICa,L after ryanodine exposure. Thus the absolute requirement for SR activator Ca2+ seen in our experiments may be uniquely dependent on the experimental conditions. Depletion of SR Ca2+ did result in larger baseline ICa,L measurements, which has also been previously reported (31) in rat and guinea pig ventricular myocytes. If ICa,L facilitation by CaM kinase does contribute to EADs, this facilitation must occur during an [Ca2+]i window that favors facilitation over inhibition.

These interventions, however, did not markedly change the pattern of the secondary inhibitory phase, indicating different underlying mechanisms. The secondary phase of ICa,L inhibition may partially reflect direct [Ca2+]i-dependent inactivation, because it was inhibited by BAPTA (Fig. 3A) and occurred at higher [Ca2+]i levels than the augmentation response (Fig. 2B). It is also possible that SR activator Ca2+- and CaM kinase-mediated ICa,L augmentation only occurs in a subpopulation of L-type Ca2+ channels but that direct Ca2+-dependent inactivation affects all L-type Ca2+ channels. The time course and magnitude of ICa,L facilitation and inhibition shown in these experiments are likely dissimilar to these processes under physiological conditions. Recent work has suggested that Ba2+ may also contribute to direct ion-dependent inactivation of ICa,L (12), and this mechanism may partially account for the decrease in Ba2+ current with pacing (Fig. 5B). Because ion-dependent inactivation of ICa,L by Ba2+ is less than with Ca2+, voltage- and/or use-dependent inactivation must also participate in the secondary inhibition of IBa,L.

CaM kinase activation transduces the augmentation of ICa,L1 and ICa,L2 by increased [Ca2+]i. The intracellular Ca2+ buffering conditions used in these experiments were chosen to give a physiological resting [Ca2+]i, which we have previously shown to be compatible with CaM kinase-mediated ICa,L augmentation (1). The amount of EGTA used in these experiments (10 mmol) was unlikely to buffer the large increases in [Ca2+]i present in the immediate vicinity of most L-type Ca2+ channels (34). The resting endogenous CaM kinase activity will be a function of [Ca2+]i and resultant levels of autophosphorylated (constitutively active) CaM kinase (6). We recently reported (2) that endogenous constitutively active CaM kinase comprises ~8% of cardiac CaM kinase under control conditions and that this amount increases to ~12% after action potential prolongation and EADs.

Complementary experimental approaches were used in the present set of experiments to demonstrate that CaM kinase activation was necessary for initial ICa,L augmentation in response to pacing. The first approach employed the use of an inhibitor peptide that completely abolished initial augmentation without changing the secondary inhibitory phase of ICa,L to pacing or altering the magnitude of ICa,L at the first beat compared with that of the control (Fig. 4B). Inhibitor peptides directed against PKA or PKC modestly reduced peak ICa,L1 and ICa,L2 augmentation but did not generally alter the biphasic response of either of these ICa,L components to pacing (Fig. 4, C and D). Although much more specific than other nonpeptide inhibitory agents, each inhibitor peptide may affect other serine/threonine kinases under some experimental conditions (28), and partial inhibition of CaM kinase could thus explain the small reductions seen in peak ICa,L1 and ICa,L2 after dialysis with the PKA and PKC inhibitor peptides (Fig. 4E).

The second approach employed the dialysis of constitutively active monomeric CaM kinase using extracellular Ba2+ (substituted for Ca2+) as the charge carrier. This approach offers the advantage that measured responses are as specific as allowed for by the kinase consensus phosphorylation sites. Abolition of increased Ba2+ current by coadministration of the CaM kinase inhibitor peptide with constitutively active CaM kinase provides evidence that the inhibitor peptide and the exogenous CaM kinase act at the same target site(s) and, thus, have similar specificities. It is known that Ba2+ does not substitute for Ca2+ to activate CaM kinase (9), and Ba2+ in the absence of constitutively active CaM kinase did not result in augmentation of IBa,L1 or IBa,L2 with pacing under our experimental conditions (Fig. 6C), suggesting that residual Ca2+ did not mediate this effect. We initially anticipated that the addition of constitutively active CaM kinase would maximally activate ICa,L and thereby obscure the stimulation-related augmentation. Instead, a pattern of stimulation-dependent ICa,L increase persisted. Because this increase was dependent on CaM kinase, it seems likely that CaM kinase facilitation of ICa,L is voltage and/or use dependent, as is the case for PKA augmentation of skeletal ICa,L (27). Dialysis of constitutively activated CaM kinase reconstituted the response of ICa,L to pacing seen under control conditions (Fig. 6B), suggesting that CaM kinase may act in a "downstream" fashion after the release of SR activator Ca2+ to augment ICa,L1 and ICa,L2.

L-type Ca2+ channels, ryanodine receptors, and CaM kinase partially colocalize in the vicinity of the T tubule. The immunofluorescent antibody colocalization experiments demonstrated that the [Ca2+]i homeostatic elements necessary for the ICa,L responses to pacing, as identified in this study, are largely restricted to the same subcellular domain, the T tubules (Fig. 5). It was previously known that L-type Ca2+ channels and ryanodine receptors colocalized at the Z band in cardiac muscle (8). In addition to the clear concentration of CaM kinase in the Z band, there is also evidence for a longitudinal pattern of CaM kinase staining in these myocytes, possibly reflecting the localization of CaM kinase with longitudinal SR, where it may regulate Ca2+ ATPase activity by phosphorylating phospholamban (25), as well as diffuse cytosolic staining. These findings, which indicate cellular localization of total CaM kinase, extend the work of Xiao et al. (33), who detected autophosphorylated CaM kinase localized to the sarcolemma and Z bands in rat ventricular myocytes. Together, these findings emphasize the importance of local CaM kinase activation. Future experiments investigating the mechanism(s) of cardiac CaM kinase localization are necessary to clarify these findings. However, the present qualitative finding adds to an important and growing body of information that the activity of multifunctional serine/threonine kinases (PKA, PKC, and CaM kinase) is made specific by tight cellular regulation of enzyme distribution. The regulation of PKA and PKC distribution within the cell is determined by specific anchoring proteins and cytoskeletal elements (13, 24). Recently, it was shown that neuronal CaM kinase can directly associate with the N-methyl-D-aspartate-type glutamate receptor Ca2+ channel (29). The identification of such anchoring proteins in cardiac tissue will further define the role of cellular targeting of these proarrhythmic kinases.

CaM kinase is a proarrhythmic signaling molecule for EADs. The CaM kinase-mediated augmentation of ICa,L1 and ICa,L2 shown in these experiments supports the hypothesis that CaM kinase is a proarrhythmic signaling molecule. The rationale of this hypothesized mechanism is that CaM kinase activity would increase due to increased [Ca2+]i after action potential prolongation and favor EADs under non-steady-state conditions by augmenting ICa,L. To test this prediction, action potentials were prolonged by slow pacing (0.1 Hz) and decreased extracellular K+ (Fig. 7). EAD induction was significantly reduced in ventricular myocytes dialyzed with the CaM kinase inhibitor peptide. Although the actions of the CaM kinase inhibitor peptide at sites other than L-type Ca2+ channels cannot be excluded as the basis for EAD suppression, our recent results showed that these peptides do not affect the transient outward current, the delayed rectifier, or the inward rectifier currents in rabbit ventricular myocytes (2). The time course of EAD induction cannot be correlated with the time course of CaM kinase-dependent ICa,L facilitation and subsequent inhibition (Figs. 1 and 4) because of the differences in experimental conditions. These results are consistent with the prediction that CaM kinase activity is proarrhythmic in its augmentation of ICa,L. Thus it is hoped that CaM kinase inhibition may be useful as an alternative or an adjunct to direct ion-channel antagonists for antiarrhythmic therapy.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Lou De Felice and Dan Roden for thoughtful comments on and criticisms of this manuscript. The CaM kinase control and inhibitor peptides were a generous gift from Dr. Howard Schulman.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant HL-03727 and a Cardiac Arrhythmia Research and Education Foundation (Irvine, CA) Grant (to M. E. Anderson) and by American Heart Association (AHA) Grant-in-Aid 96010040 (to R. J. Colbran). R. J. Colbran is an AHA Established Investigator. L. B. MacMillan is supported by Fellowship No. 97F101 from the Tennessee Affiliate, AHA. Immunofluorescence staining experiments were performed in part through use of the Vanderbilt University Medical Center Cell Imaging Resource (supported by National Institutes of Health Grants CA-68485 and DK-20593).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. E. Anderson, Vanderbilt University Medical Center, 315 Medical Research Building II, Nashville, TN 37232-6300 (E-mail: mark.anderson{at}mcmail.vanderbilt.edu).

Received 21 September 1998; accepted in final form 22 February 1999.


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DISCUSSION
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Am J Physiol Heart Circ Physiol 276(6):H2168-H2178
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