Abstract
The multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) mediates Ca++-induced augmentation of L-type Ca++ current (ICa); therefore it may act as a proarrhythmic signaling molecule during early afterdepolarizations (EADs) due to ICa. To investigate the hypothesis that ICa-dependent EADs are favored by CaM kinase activation EADs were induced with clofilium in isolated rabbit hearts. All EADs were rapidly terminated with ICaantagonists. Hearts were pretreated with the CaM kinase inhibitor KN-93 or the inactive analog KN-92 (0.5 μM) for 10 min before clofilium exposure. EADs were significantly suppressed by KN-93 (EADs present in 4/10 hearts) compared to KN-92 (EADs present in 10/11 hearts) (P = .024). There were no significant differences in parameters favoring EADs such as monophasic action potential duration or heart rate in KN-93- or KN-92-treated hearts. CaM kinase activity in situincreased 37% in hearts with EADs compared to hearts without EADs (P = .015). This increase in CaM kinase activity was prevented by pretreatment with KN-93. In vitro, KN-93 potently inhibited rabbit myocardial CaM kinase activity (calculatedKi ≤ 2.58 μM), but the inactive analog KN-92 did not (Ki > 100 μM). The actions of KN-93 and KN-92 on ICa and other repolarizing K+currents did not explain preferential EAD suppression by KN-93. These data show a novel association between CaM kinase activation and EADs and are consistent with the hypothesis that the ICa and CaM kinase activation both contribute to EADs in this model.
EADs are depolarizing oscillations in the AP that occur during repolarization (January and Moscucci, 1992). One cause of EADs is the reactivation of L-type ICa within a range, or window, of Vm over which the steady-state inactivation and activation relationships of ICa overlap (January and Riddle, 1989). Prolongation of AP repolarization increases the time that Vm is in this window current range for ICa and thus increases the likelihood of ICa reactivation and the induction of EADs. EADs are clinically important because they are the probable initiating cause of lethal arrhythmias associated with long QT intervals including torsade de pointes (Roden et al., 1996). A long QT interval reflects prolonged AP repolarization in ventricular myocardium and is due to a wide variety of conditions including bradycardia and hypokalemia (Roden et al., 1996). One important cause of long QT intervals are antiarrhythmic drugs and the ventricular proarrhythmic effects of many antiarrhythmic agents are due to QT interval prolongation (Roden et al., 1996).
[Ca++]i increases during AP prolongation (Bouchard et al., 1995) and EADs in isolated ventricular myocytes (De Ferrari et al., 1995). Elevation of [Ca++]i has complex effects on ICa including indirect enhancement through a multifunctional Ca++/calmodulin-dependent protein kinase II (CaM kinase) pathway (Xiao et al., 1994; Yuan and Bers, 1994; Anderson et al., 1994) and direct inactivation (de Leon et al., 1995). We have previously shown that the net effect of elevated [Ca++]i in rabbit ventricular myocytes after flash photolysis of the photolabile Ca++ chelator Nitr-5 is 40 to 50% augmentation of peak ICa that is mediated by CaM kinase (Anderson et al., 1994). CaM kinase is an ubiquitous serine/threonine protein kinase that is activated by Ca++-bound calmodulin (Braun and Schulman, 1995a). Once activated by Ca++/calmodulin, CaM kinase activity can be sustained by intersubunit enzyme autophosphorylation that confers Ca++-independent activity (Braun and Schulman, 1995a), allowing for CaM kinase activity to persist during the long diastolic intervals seen with QT interval prolongation associated ventricular arrhythmias such as torsade de pointes. This further suggests the hypothesis, tested here, that EADs caused by ICa may be enhanced by increased [Ca++]i through this CaM kinase pathway.
KN-93 is a recently synthesized methoxybenzene sulfonyl derivative with enhanced water solubility that competitively inhibits calmodulin binding to CaM kinase with a reported Ki of 0.37 μM (Sumi et al., 1991). KN-93 has been shown to inhibit CaM kinase-dependent processes in PC12h cells (Sumi et al., 1991), fibroblasts (Tombes et al., 1995) and gastric parietal cells (Mamiya et al., 1993). The catalytic and regulatory domains in CaM kinase are highly conserved in all known CaM kinase isoforms, so inhibitors that interact with either of these domains are expected to exert this effect in all cell types including cardiac myocytes (Braun and Schulman, 1995a). KN-92 is a congener of KN-93 without CaM kinase inhibitory activity and is used as an experimental control (Tombes et al., 1995). Neither KN-93 or KN-92 have appreciable effects on other serine/threonine kinases such as protein kinase A (PKA) or protein kinase C (PKC) (Niki et al., 1993; Sumi et al., 1991). In addition to CaM kinase inhibitory activity, KN-93 (and KN-92), as with other CaM kinase and calmodulin inhibitors (Braun and Schulman, 1995a) may be a direct ICa antagonist (Li et al., 1997).
Although EADs can occur in all cardiac cell types, specific cardiac cells may be especially important for generation of EADs. Both Purkinje fibers (El-Sherif et al., 1988) and cells in the ventricular midmyocardium called M cells (Weirich et al., 1996; Liu and Antzelevitch, 1995) develop markedly prolonged AP durations at slow heart rates relative to most myocytes and are particularly prone to develop EADs. Studies in isolated myocytes or superfused isolated tissue preparations thus may lack important cell groups for the generation and propagation of EADs. Hence, studies in the isolated heart have the advantage of containing all these cell types and so may allow for more physiologically relevant information regarding EAD induction and suppression than similar studies in isolated cells.
In this study we used the novel cell membrane permeant CaM kinase inhibitor KN-93 as a tool for testing the hypothesis that EADs are associated with CaM kinase activation. We present evidence that EADs in this model are associated with enhanced CaM kinase activity, dependent on ICa, and suppressed by KN-93. These findings are thus consistent with the hypothesis that CaM kinase is a proarrhythmic signaling molecule for EADs in this isolated heart model.
Methods
Langendorff preparation.
New Zealand White rabbits of either gender (2.7–3.2 kg) were first given a bolus injection with heparin (150 U/kg i.v.) and killed by pentobarbital (50 mg/kg i.v.) overdose. Hearts were rapidly excised immediately after the withdrawal reflex was no longer present and placed in ice-cold Tyrode’s solution (see below) for dissection of extracardiac tissue. The aorta was cannulated, and perfused retrograde with Tyrode’s solution. The perfusate (200 ml) was recirculated through a warming bath and filling pressure was adjusted to 20 cm of H2O. Recirculation time was ∼1 min, as judged by the onset of decreased LV pressure after addition of ICablockers to the recirculating perfusate. Perfusate temperature was monitored using a thermistor probe positioned in the RV and maintained at 33 ± 1°C by a closed loop feed back system. The perfusate was vigorously bubbled with 95% O2, 5% CO2and pH was monitored throughout the experiments and adjusted to 7.4 with 1 N NaOH or HCl, as appropriate. Both atria were removed and drains were placed in the RV and LV apices. Leads for recording an ECG were sutured to the LV apex and the proximal pulmonary artery. Epicardial pacing wires were inserted into the RV free wall. Bradycardia was produced by crushing the AV node with forceps. Epicardial pacing was used to maintain a minimum cycle length of 1700 to 1500 msec (heart rate of 35–40 beats/min). Pacing output was adjusted to twice diastolic threshold. In most experiments (see below) LV pressure was continuously monitored with a solid state transducer placed in a saline filled latex balloon. The balloon was secured with a purse string suture around the left atrial remnant and pressure was adjusted to maximize the LVDP keeping the LV end diastolic pressure <10 mmHg. LVDP (peak systolic pressure - peak diastolic pressure) measurements are reported as the mean of three consecutive beats except during irregular rhythms and at EAD termination. During irregular rhythms, LVDP measurement is reported as the mean of three beats following the longest interbeat intervals. After ICablockade, LVDP is reported as the first beat without an EAD (fig.1C). LVDP stability was defined as <10% interbeat variability. All procedures involving animals were in accordance with institutional guidelines.
Preparation of single ventricular myocytes.
Single ventricular myocytes were prepared as previously described (Andersonet al., 1994). Briefly, New Zealand White rabbits (2–3 kg body weight) were killed by pentobarbital (50 mg/kg i.v.) overdose. Hearts were rapidly excised and placed in ice cold nominally Ca++ free HEPES-buffered myocyte bath solution (see below). The aorta was cannulated, and the heart was perfused in a retrograde fashion with a nominally Ca++ free perfusate for 15 min at 37°C. This was followed by a 15-min perfusion with collagenase-containing nominally Ca++ free solution. Final perfusion was with collagenase-containing low Ca++ (0.2 mM) solution. The LV and septum were cut away, coarsely minced and placed in a beaker containing low Ca++ solution with 1% (w/v) BSA at 37°C. Myocytes were dispersed by gentle agitation, collected in serial aliquots and then maintained in standard saline solution containing 1.8 mM Ca++. All solutions were vigorously oxygenated.
Measurement of CaM kinase activity in vitro.
Left ventricular tissue homogenate was prepared as follows: 1 to 2 g of the left ventricular free wall was quickly cut away from the perfused, beating heart, coarsely minced and suspended in 5 to 10 ml of ice-cold homogenization buffer (mM: 20 PIPES, 1 EDTA, 1 EGTA, 2 DTT, 10 sodium pyrophosphate and 10–50 μg/ml leupeptin) at pH 7.0. Tissue suspension was homogenized at 4°C using 3- to 10-sec bursts of a Polytron with 30-sec pauses in between bursts. The homogenate was then centrifuged at ∼10,000 × g for 20 min at 4°C using a JA-20 rotor. The supernatant fraction was removed and used directly in the assay. Protein concentration was measured by the method ofBradford (1976), using BSA as the standard.
Assay of CaM kinase activity was performed essentially as described byWaldmann et al. (1990) with minor modifications. The assay was performed in triplicate in a final volume of 50 μl containing 50 mM PIPES, pH 7.0, 10 mM MgCl2, 0.1 mg/ml BSA, 10 μM autocamtide-3 (selective CaM kinase peptide substrate), 150 nM calmodulin, 1 mM CaCl2 or 1 mM EGTA, and increasing concentrations (0–100 μM) of KN-93 or KN-92. A total of 30 to 50 μg of tissue homogenate protein was added per assay tube, and samples were then preincubated at 30°C for 1 min. The kinase reaction was started by addition of 50 μM (final) γ32P-ATP (∼400 cpm/pmol), and incubation was carried out for an additional 1 to 2 min. The reaction was terminated by addition of 10 μl cold trichloroacetic acid (30% w/v), and samples were then placed on ice for ≥2 min. Ca++/calmodulin-dependent phosphorylation of the autocamtide-3 substrate was quantified as described (Waldman et al., 1990). Ca++/calmodulin-independent CaM kinase activity was assayed as described above, but Ca++ was replaced in the assay mixture by EGTA.
In experiments quantifying Ca++-independent CaM kinase activity from perfused beating hearts, the same criteria for LVDP and MAP stability were applied to all hearts prior to experimental interventions. Control conditions for the isolated hearts were established and the in vitro assay for Ca++-independent CaM kinase activity was performed as described above. Addition of clofilium (7.5 μM) and pretreatment with KN-93 (0.5 μM) as well as criteria for EAD induction were as described below. A stable pattern of EADs was present for 10 min before obtaining LV tissue from the clofilium-treated hearts for the purpose of the in vitro kinase assay. For assays specifically measuring the dose-dependent inhibition of CaM kinase by KN-93 and KN-92, LV homogenates were prepared from hearts immediately after removal, with no prior perfusion or treatments.
Data describing inhibition of CaM kinase activity by KN-93 were fit according to the Hill equation:
Solutions.
Tyrode’s solution was composed of (mM) NaCl 130.0, NaHCO3 20.0, glucose 5.6, KCl 3.0, CaCl22.0, NaH2PO4 1.8, MgCl24H2O 0.7. All drugs were added to the recirculating perfusate to yield final reported concentrations: (μM) Clofilium (Research Biochemicals International, Natick, MA) 7.5, KN-92 0.5, KN-93 0.5 (Seikagaku America, Ijamsville, MD), nifedipine 10.0 and Cd++ 200–400. Nifedipine and KN-92 were dissolved in DMSO. The maximum final DMSO concentration was 10−3 vol %, which had no effect on ICa or IK in voltage clamp studies. Unless otherwise mentioned all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
The standard Ca++ containing solution for myocyte preparation was composed of (mM) NaCl 137.0, HEPES (free acid) 10.0, NaH2PO4 0.33, glucose 10.0, KCl 5.4, CaCl2 1.8 and MgCl2 2.0. The nominally Ca++ free solution was identical, except CaCl2was omitted. The low Ca++ solution contained 0.2 mM CaCl2. The collagenase-containing solutions were prepared with 1% BSA (w/v) and ∼60 U/ml type I collagenase (Worthington Biochemicals, Lakewood, NJ) and ∼0.1 U/ml type XIV protease.
The myocyte bath solution for recording ICa contained (mM) choline Cl or N-methyl-d-glucamine 137, HEPES 10, KCl 5.4, CaCl2 1.8, MgCl2 0.5, CsCl 20 or 25. The pH was adjusted to 7.4 with 1 N CsOH. For recordings of ICa, micropipettes were filled with an intracellular solution containing (mM) CsCl 120, CaCl2 1.0, TEA 10, NaGTP 1.0, Na2 phosphocreatine 5.0, HEPES 10, EGTA 3.0, Cs4BAPTA 20. The pH was adjusted to 7.2 with 1 N CsOH. Myocyte bath solutions for recording IK contained (mM) N-methyl-d-glucamine 149, HEPES 5.0, glucose 5.0, KCl 1.0, MgCl2 5.0. The pH was adjusted to 7.4 with 10 N HCl. Osmolality was measured for all solutions and found to be 280 to 300 mOsm after adjusting the pH. 4-AP (4 mM) and niflumic acid (10 μM) were included in the bath solution for some experiments. Dofetilide (2 μM) was a generous gift from Pfizer Central Research (Sandwich, U.K.). For recordings of IK micropipettes were filled with an intracellular solution containing (mM) K aspartate 120, HEPES 5.0, KCl 25, Na2ATP 4.0, MgCl2 1.0, K4BAPTA 0.1, Na2 phosphocreatine 2.0, Na GTP 2.0. The pH was adjusted to 7.2 with 1 N KOH.
CaM kinase inhibitory peptide.
A peptide CaM kinase inhibitory peptide, AC3-I, (KKALHRQEAVDCL) was included in the pipette solution at a final concentration of 20 μM for some experiments. This amino acid sequence HRQEA corresponds to the autophosphorylation site on CaM kinase (Braun and Schulman, 1995b) except the T (Thr 286 of α CaM kinase) was modified to A (Ala) to prevent phosphorylation. Thus, AC3-I is a modified CaM kinase substrate that has an IC50∼3 μM for CaM kinase activity in vitro (Braun and Schulman, 1995b). Importantly, AC3-I has little inhibitory activity against other serine threonine kinases (e.g., IC50 ∼500 μM for protein kinase C-dependent substrate phosphorylation) (Braun and Schulman, 1995a,b). Such inhibitors prevent interaction of the activated CaM kinase with potential substrates (Braun and Schulman, 1995a,b). The inhibitor peptide was prepared using a solid-phase peptide synthesizer (Applied Biosystems, Foster City, CA) and purified by reverse-phase high-performance liquid chromatography. The sequence was confirmed by automated sequencing.
MAP measurements.
In most experiments a single MAP electrode (EP technologies Inc., Sunnyvale, CA) was positioned over the LV epicardium with a macro-manipulator. The heart was positioned against an adjustable stop opposite the epicardial MAP catheter to minimize heart motion. In a preliminary series of experiments designed to test the concordance of endocardial and epicardial EADs the LV balloon was omitted and paired MAP catheters were positioned opposite one another on the LV epicardial and endocardial free wall. MAP recordings were concordant for the presence (n = 8) and absence (n = 4) of EADs in all but one instance, suggesting that epicardial MAP recording is a valid method for detecting EADs from LV epicardium and endocardium in this isolated heart model. All MAP signals were amplified from 0 to 100 Hz with a direct current coupled preamplifier (EP technologies Inc., Sunnyvale, CA) and recorded directly onto a chart recorder (Gould 2600S) at 100 mm/sec. Data from some experiments were digitized (Neuro-corder) and stored on a videotape recorder. Acceptable MAP catheter position was confirmed by a stable action potential configuration free of EADs or DADs during a >10-min control period. The MAP amplitude was defined as the difference between plateau (phase 2) and diastole (phase 4). MAP amplitude at the start of the experiments was 10.4 (±1.26) mV. The MAP duration was measured from the onset of phase 0 to the point of 50% (APD50) and 90% (APD90) repolarization. When present (see below), EADs were included in the APD measurements. All MAP durations are reported as the mean of three consecutive beats, except after EAD termination where the first beat after EAD termination by Cd++ or nifedipine was used (fig. 1C). Beat to beat intervals were measured from the onset of phase 0 using contiguous MAPs. Beat to beat intervals during irregular rhythms are reported as the longest intervals present during the measurement period.
EAD definition, induction and experimental design.
EADs were defined as discrete oscillations in MAP repolarization with a positive slope of the tangent to the onset of the oscillation (fig. 2, A and C). Experimentally, EADs had to be present during >90% beats over a 1-min period or in a stable bigeminal alternating pattern to be classified as inducible. Once these criteria were met we found that EADs continued for >2 min and always required ICa blockade for termination. Multiple EADs were defined as >1 EAD occurring per beat (fig. 1A). The CaM kinase inhibition experiments consisted of 4 periods (fig. 1, A and B). The first period (>10 min) was the control and established stable baseline values for MAPs, LVDP, heart rate and rhythm. The second period followed addition of KN-92 (0.5 μM) or KN-93 (0.5 μM) to the perfusate and lasted 10 min. The third period followed addition of clofilium (7.5 μM) to the perfusate and lasted for 30 min or until EADs were observed. If no EADs occurred, then the longest MAP durations were measured. MAP and LVDP measurements were made at 10-min intervals and at the time of EAD occurrence during the third period. A fourth period was only used in experiments where EADs had occurred, and measurements followed addition of nifedipine (10 μM) or Cd++ (200–500 μM) to the perfusate.
Quantitation of Ca++-independent CaM kinase activity was performed on LV homogenate from isolated perfused rabbit hearts. Control conditions for the isolated hearts were established and thein vitro assay for Ca++-independent CaM kinase activity was performed as described above. Addition of clofilium (7.5 μM) and pretreatment with KN-93 (0.5 μM) as well as criteria for EAD induction were as described above. A stable pattern of EADs was present for 10 min before obtaining LV tissue from the clofilium-treated hearts for the purpose of the in vitroassay.
Voltage clamp measurements.
Isolated quiescent ventricular myocytes were studied with patch-clamp methodology in the whole cell recording configuration (Hamill et al., 1981) using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Micropipettes were pulled from glass capillary tubing (VWR) and heat-polished to a tip resistance of 1 to 3 MΩ when filled with the intracellular solution. Voltage clamp protocols and data acquisition were performed using PCLAMP software (version 6.0, Axon Instruments) on a microcomputer with a A/D, D/A converter (Digidata 1200, Axon Instruments). Transmembrane current was sampled at 5.56 kHz. All ICa measurements were performed at room temperature (20–23°C) on the stage of a Diaphot inverted microscope (Nikon). The IK measurements were performed at 33–36°C using a heated stage recording chamber (Warner Instrument Corp., Grand Haven, MI).
For ICa measurements cell membrane potential was held at −80 mV and pulsed to potentials from −40 mV to +50 mV for 300 msec in 10 mV steps. The interpulse interval was 1500 msec. Time dependent recovery of ICa from inactivation was measured using a double pulse protocol by inserting progressively longer interpulse intervals (P1 to P2) from a holding potential of −80 mV to a test Vm of 0 mV for 300 msec. Intracellular Ca++ buffering with BAPTA (20 mM) was chosen to minimize CaM kinase activation allowing direct ICa blockade by KN-93 or KN-92 to be measured, independent of a drug effect on CaM kinase. KN-92 or KN-93 was added to the bath at increasing concentrations for dose response determinations.
Effects of KN-92 and KN-93 on IK were assessed by measuring the Vm dependence of peak tail current amplitudes. Values were normalized to the maximum peak tail current in the absence of KN-92 or KN-93. Half maximal activation voltage (V1/2) and the slope factor were determined by fitting the normalized data to a Boltzmann function: I/Imax = (1 + exp [(V1/2 − Vt)/k])−1, where Imax is the peak tail current, Vt is the conditioning Vm and k is the slope factor. Conditioning pulses of 440 msec (in the range of MAP APD50) applied every 1500 msec were used to approximate the range of measured MAP durations. A holding potential of −80 mV was used prior to the conditioning voltage step. A brief (20 msec) step to −40 mV was applied before the conditioning pulse to inactivate Na+ current (INa). Tail currents were induced by stepping to −40 mV from the conditioning voltage step. In a second set of experiments a −40 mV holding potential was used to inactivate INa before 1000 msec conditioning pulses to check for slow components of IK.
Data analysis.
Data are presented as mean ± S.E.M. P values were assessed with analysis of variance or paired t test as appropriate except for EAD induction rates where the Fisher exact test was used. Statistically significant differences were defined as P < .05.
Results
Clofilium-induced early afterdepolarizations are terminated by L-type Ca++ current antagonists.
The experimental design for the isolated heart experiments is shown in figures 1, A and B. All clofilium-induced EADs were terminated by the ICaantagonists Cd++ (200–500 μM, n = 4) or nifedipine (10 μM, n = 7). EAD termination occurred within 10 sec from a discernible decrease (>10% from preceding baseline) in LVDP after addition of ICa antagonist to the perfusate (fig. 1C). EAD termination occurred before LVDP decreased significantly and without a change in the interbeat interval (fig. 2). APD was not shortened upon the first beat of EAD termination (fig.3). EAD termination by ICaantagonists was unlikely due to a secondary effect on [Ca++]i because LVDP was not significantly depressed at initial termination. Thus, EAD termination by ICa antagonists was not due to secondary effect on MAP duration or heart rate, thereby supporting the hypothesis that ICa is an important source of inward current for clofilium-induced EADs in rabbit myocardium.
Early afterdepolarizations are prevented by pretreatment with the CaM kinase inhibitor KN-93.
We next examined the hypothesis that CaM kinase may contribute to ventricular EAD initiation and maintenance, given its role in the enhancement of ICa (Xiaoet al., 1994; Yuan and Bers, 1994; Anderson et al., 1994). Isolated hearts studied as in figure 1 and pretreated with the CaM kinase inhibitor KN-93 (0.5 μM) were significantly less likely to develop EADs in response to clofilium (7.5 μM) (EADs in 4/10 hearts) than hearts pretreated with the inactive analog KN-92 (0.5 μM) (EADs in 10/11 hearts) (summarized in table1). EAD induction by clofilium was not different in hearts pretreated with KN-92 compared to hearts without any type of pretreatment (EADs in 8/8 hearts), suggesting that the inactive analog KN-92 had no effect on EAD inducibility by clofilium at the concentration used in this study. When EADs were subclassified as single or multiple oscillations during MAP repolarization, multiple EADs occurred more frequently in hearts pretreated with the inactive analog KN-92 (7/11) compared with those pretreated with the CaM kinase inhibitor KN-93 (1/10) (P = .024) (table 1). The differences in EAD inducibility in hearts pretreated with KN-93 and KN-92 were not due to differences in heart rate (fig. 2) or MAP duration (fig. 3). These findings suggest that KN-93 suppression of EADs is independent of factors that are known to facilitate EADs, such as heart rate and action potential duration.
KN-93 prevented increased LV developed pressure during action potential prolongation and early afterdepolarizations.
LV developed pressure increased significantly (P = .014), compared with control, following addition of clofilium to the bath in hearts pretreated with the inactive analog KN-92 (fig. 2). Left ventricular developed pressure increases occurred in step with MAP prolongation (fig. 3) and EADs. KN-93 pretreatment completely prevented the increase in LVDP by clofilium (fig. 2) without reducing MAP duration (fig. 3). These data suggest that increased LVDP during MAP prolongation and EADs may be due to (a) CaM kinase-mediated process(es).
Ca++-independent CaM kinase activity is increased during early afterdepolarizations and this increase is prevented by KN-93.
After activation by increased [Ca++]i, CaM kinase activity becomes partially Ca++-independent by a mechanism in which normal Ca++/calmodulin-dependent activation leads to autonomy via intersubunit autophosphorylation (Braun and Schulman, 1995a). Thus, Ca++-independent activity serves as a marker for the magnitude of in situ CaM kinase activation by [Ca++]. Because ICa is important for EADs in this isolated heart model, we hypothesized that EADs are associated with increased CaM kinase activity. Ca++-independent CaM kinase activity increased significantly (P = .015) in hearts demonstrating EADs after clofilium (n = 5) compared to hearts without clofilium treatment or EADs (n = 5) (fig. 4). However, no significant increase in Ca++-independent CaM kinase activity was observed in hearts pretreated with KN-93 (0.5 μM) prior to clofilium addition (P = .461, compared to control, n = 6). These findings indicate that EADs are associated with an increase in myocardial CaM kinase activity and that the concentration of KN-93 used in these experiments is sufficient to significantly inhibit CaM kinase activation. These findings show an association between EADs and CaM kinase activation and are consistent with the hypothesis that CaM kinase activation may favor EADs and CaM kinase inhibition may suppress EADs.
KN-93 inhibits CaM kinase II activity in rabbit myocardium.
In vitro measurements of CaM kinase activity in LV tissue homogenate show that KN-93 is a potent inhibitor of CaM kinase with a calculated Ki = 2.58 μM (fig.5). This value is an upper estimate because the calculation does not take into account free endogenous calmodulin present in the tissue homogenate. The inactive analog KN-92 did not cause appreciable inhibition of CaM kinase activity. Similar findings have been previously reported in rat pheochromocytoma (PC12) cells (Sumi et al., 1991). The present results show that KN-93, but not KN-92, inhibits rabbit myocardial CaM kinase activity at a concentration comparable to that used to inhibit EADs in the isolated heart experiments.
Direct L-type Ca++ current antagonist action of KN-92 and KN-93.
Based on the observation that clofilium-induced EADs are terminated by ICa blockade in isolated rabbit hearts (fig. 1C), along with findings that other cell membrane permeant CaM kinase inhibitory agents block ICa (Li et al., 1992; Klockner and Isenberg, 1987; Anderson et al., 1994), we assessed the relative ICa blocking potency of KN-92 and KN-93 in single rabbit left ventricular myocytes. Because CaM kinase is known to enhance ICa, we blocked CaM kinase activation by dialyzing cells, via the recording micropipette, with the Ca++ chelator BAPTA (20 mM), so that only direct effects of KN-92 and KN-93 on ICa could be measured. External application of either KN-92 (0.5 μM) or KN-93 (0.5 μM) each caused a modest steady state inhibition of peak ICa (7 ± 6.0%, KN-93; 10 ± 4.6%, KN-92) (P = .702) in single ventricular myocytes (fig. 6, A–C). However, at a higher concentration (1.0 μM) KN-93 more potently inhibited peak steady state ICa (41 ± 7%) compared to KN-92 (13 ± 10%, P = .005), consistent with a recently published report (fig. 6C) (Li et al., 1997). Neither KN-93 or KN-92 shifted the apparent voltage dependence of ICaactivation (fig. 6, A and B) or affected time dependence of recovery from inactivation (fig. 6D) at the concentration used in the isolated heart preparation (0.5 μM). The time constants (τ) for recovery from inactivation were not different for control cells (113 msec), or in cells treated with 0.5 μM KN-93 (98 msec) or KN-92 (109 msec). These results suggest that at the concentration used in this study to suppress EADs in isolated, beating hearts, KN-93 and KN-92 are equipotent as direct blockers of ICa. Therefore, the greater EAD suppression by KN-93 compared with KN-92 cannot be due to direct ICa blockade.
KN-93 directly inhibits the delayed rectifier, the inward rectifier and the transient outward potassium currents.
Because EAD induction in this model is dependent on the IK antagonist clofilium, we tested the possibility that enhancement of repolarizing current by KN-93 (or reduction of repolarizing current by KN-92) could result in the relative reduction in EADs observed in KN-93 treated hearts (table 1), independent of CaM kinase inhibition. However, IK tail currents were significantly inhibited by KN-93 but not by KN-92 (fig. 7, A and B). The V1/2 modestly shifted in the hyperpolarizing direction (14.6 mV to 2.3 mV) after KN-93. These tail currents were predominantly the rapid component of IK (IKr) as evidenced by the fact that they were >95% inhibited by the IKrantagonist dofetilide (2.0 μM, n = 6) at all conditioning voltages (data not shown). To address whether this antagonist action of KN-93 was due to CaM kinase inhibition or a direct action on the ion channel, single myocytes were dialyzed via the recording micropipette with the specific CaM kinase inhibitory peptide AC3-I (see “Methods”). IKr was found to be unchanged after dialysis with AC3-I, suggesting that inhibition of CaM kinase is unlikely to account for the observed blockade of IKr by KN-93 (fig. 7C). A second set of experiments was performed using longer (1000 msec) conditioning pulses to examine whether KN-92 or KN-93 affected the slower components of IK. KN-93, but not KN-92, (both at 0.5 μM) significantly inhibited IK after conditioning pulses positive to +10 mV (fig. 7D) and modestly shifted V1/2 in a hyperpolarized direction (7.3 to −4.8 mV). Because inhibition of IK by KN-93 is anticipated to favor EAD induction (Roden et al., 1996), the lower EAD inducibility observed in KN-93-treated hearts (table 1) cannot be explained by differential actions of KN-93 on IK.
Two other outward currents were elicited by the voltage clamp protocol used for measuring IK tails (fig. 7A). A prominent early outward current with rapid activation (time to peak within 10 msec) and inactivation (100–200 msec) was present during the conditioning voltage steps. This current is at least partly Ito as peak early outward current was significantly inhibited by 4-AP (4 mM) at test voltages positive to −20 mV (data not shown, n = 6) (Hiraoka and Kawano, 1989). Subsequent addition of the Cl− current antagonist niflumic acid (10 μM) did not further reduce this current (data not shown, n = 3). KN-93, but not KN-92, (both at 0.5 μM) significantly inhibited peak Ito at test voltages positive to −20 mV (fig. 7E). Peak Ito was not inhibited by intracellular dialysis with the CaM kinase inhibitor peptide AC3-I (data not shown, n = 8), suggesting that inhibition by KN-93 was independent of an effect on CaM kinase activity. An outward current was present at the holding potential (−80 mV) during control conditions (fig. 7A), consistent with the inward rectifier (IK1) (Giles and Imaizumi, 1988). This current was found to be blocked by 0.5 μM KN-93 (137 ± 11 control to 57 ± 17 pA, P = .002, n = 5), but not by 0.5 μM KN-92 (189 ± 30 control to 160 ± 45 pA, P = .45, n = 4) inhibited this current. KN-93 actions were likely independent of CaM kinase inhibition because IK1 was not altered after dialysis with the CaM kinase inhibitor peptide AC3-I (n = 8).
The observed blockade of these repolarizing currents by KN-93 should favor EAD induction in the perfused, beating heart preparation, which is contrary to the marked reduction in EADs seen after treatment with this agent (table 1). Thus, these findings suggest that the critical action of KN-93 in preventing the onset of EADs in the isolated heart model is inhibition of CaM kinase.
Discussion
L-type Ca++ current is important for early afterdepolarizations.
Our findings support the idea that ICa is important for clofilium-induced EADs in this isolated rabbit heart model. The evidence for ICainvolvement in these EADs is that they are terminated by specific ICa antagonists. EAD termination by ICaantagonists appears to be a direct effect of ICa blockade, and not a secondary effect of [Ca++]idepletion, because of its 1) rapid onset (fig. 1C) and 2) occurrence before significant loss of LVDP (fig. 2A). The lack of significant MAP shortening at the point of EAD termination (fig. 3) is further evidence that EAD termination is a direct effect of ICa blockade and not due to a secondary effect on MAP duration.
Early afterdepolarizations are suppressed by KN-93.
These results also show that the CaM kinase inhibitory drug KN-93, but not its inactive analog, KN-92, significantly decreases the inducibility of EADs in the whole heart model (table 1). EAD suppression by KN-93 was independent of other factors known to promote EADs. Action potential duration was not significantly different in hearts pretreated with KN-92 or KN-93 before or after clofilium (fig. 3, experimental periods 2 and 3). It may be that the negative effects of KN-93-dependent CaM kinase inhibition on ICa were balanced by a modest, direct blockade of repolarizing K+ currents, leaving the action potential duration unchanged in hearts treated with KN-93. It is clear that KN-93 has actions other than CaM kinase inhibition and this lack of specificity is a limitation of this study.
KN-93 inhibits L-type Ca++ current independent of CaM kinase inhibition.
We observed that KN-93 and KN-92 were equipotent ICa inhibitors at the concentration (0.5 μM) used in the isolated heart experiments (fig. 6). However, at 1.0 μM, under conditions adverse to CaM kinase activation, KN-93 directly inhibited ICa more potently than KN-92 (fig. 6). One possible explanation for the differential effect on EAD suppression by the two agents may be that the amount of ICa inhibition by KN-92 and KN-93 in working myocytes of the isolated heart is different (i.e., KN-93 produces stronger block at 0.5 μM) compared to isolated myocytes under voltage clamp. The observation that LVDP does not decrease relative to control values in the presence of 0.5 μM KN-92 or KN-93 (fig. 2A) is not likely consistent with profound ICa inhibition in the isolated heart experiments. Thus, reduction in EAD inducibility by KN-93 vs. KN-92 is unlikely to be due to differential direct inhibition of ICa by the two agents.
KN-93 inhibits repolarizing currents independent of CaM kinase inhibition.
KN-93, but not KN-92, does inhibit IK, Ito and IK1 at the concentration (0.5 μM) observed to suppress EADs in the isolated, beating hearts (fig. 7, A, B, D and E). Intracellular dialysis with the specific CaM kinase inhibitory peptide AC3-I (fig. 7C, data not shown for Itoand IK1) did not alter these currents, suggesting that blockade produced by KN-93 was independent of an effect on CaM kinase activity. Although the rapid component of IK(IKr) is predominant in rabbit ventricle (Carmeliet, 1992), the work of Salata et al. (1996) suggests that IKs may also be present. Inhibition of IK after short (fig. 7B) and long (fig. 7D) conditioning steps (over durations relevant to the MAP recordings in this study) was similar in response to KN-93. Inhibition of these repolarizing currents is expected to increase EAD inducibility (Roden et al., 1996) but instead a significant reduction in EAD inducibility was observed in this study (table 1). Thus observed effects of KN-93 on IK or other repolarizing currents cannot explain the reduction in EADs seen in this study.
Increased CaM kinase activation during early afterdepolarizations is prevented by KN-93.
The clofilium-induced increase in action potential duration is expected to increase Ca++ entry via ICa (Clark et al., 1996). A prolonged biphasic calcium transient is present during EADs (Miura et al., 1993; De Ferrari et al., 1995) and may enhance CaM kinase activation by Ca++/calmodulin. In addition to mediating Ca++-dependent augmentation of ICa (Xiaoet al., 1994; Yuan and Bers, 1994; Anderson et al., 1994), CaM kinase facilitates Ca++ uptake (Reddyet al., 1996; Odermatt et al., 1996) and release (Hain et al., 1995) by the sarcoplasmic reticulum. The anticipated net effect of CaM kinase activation in ventricular myocytes is increased LVDP. Clofilium did increase LVDP in KN-92, but not KN-93, pretreated hearts (fig. 2A, experimental period 3) despite equivalent MAP prolongation in KN-92 and KN-93 treated hearts (fig. 3). Once activated by Ca++/calmodulin, CaM kinase undergoes intersubunit autophosphorylation, resulting in a Ca++-independent form of CaM kinase that can remain active at low levels of diastolic [Ca++]i (Braun and Schulman, 1995a). In vitro measurement of Ca++-independent CaM kinase activity thus provides an important measure of CaM kinase activation in situ. We found that Ca++-independent activity is significantly increased during EADs and this increase is prevented by pretreatment with the CaM kinase inhibitor KN-93 (fig. 4). It is likely that the global increase in CaM kinase activity measured here reflects larger increases in CaM kinase activity occurring in certain subcellular domains. These results show an association between EADs and increased CaM kinase activity and suppression of EADs and CaM kinase inhibition. These associations are consistent with the hypothesis that increased CaM kinase activation may be proarrhythmic during action potential prolongation.
L-Type Ca++ current and intracellular Ca++in early afterdepolarizations.
The role of ICa and [Ca++]i in EADs is controversial. Earlier studies have suggested that elevated [Ca++]iin some models may not be strictly necessary for EADs due to ICa. Marban et al. (1987) found that EADs induced by external Cs+ were terminated by the ICa antagonist nitrendipine in isolated ferret papillary muscles treated with the sarcoplasmic reticulum Ca++release channel blocker ryanodine. Developed tension was markedly decreased, indicating decreased [Ca++]i at the time of EADs. Still, EADs could be inhibited by the ICaantagonist nitrendipine. However, other workers have found that treatment with agents expected to reduce [Ca++]i, such as ryanodine, do inhibit EAD formation (Priori and Corr, 1990). Carlsson et al. (1996)recently reported that the ICa antagonist nisoldipine acutely suppressed and pretreatment with ryanodine or the [Ca++]i chelator BAPTA-AM partially prevented torsade de pointes in a rabbit model. Thus, the role of ICaand [Ca++]i in EAD induction may be model dependent. However, most findings suggest a role for ICaand [Ca++]i in EADs.
Monophasic action potential recordings.
Monophasic action potential recordings do not report cell membrane potentials and are thus not identical to true transmembrane action potential recordings (Franz, 1994). Nevertheless, MAP recording has been shown to accurately depict EADs and action potential duration when compared with simultaneous microelectrode recordings (Ino et al., 1988;Franz, 1994) and EADs have been demonstrated with both techniques in rabbit myocardium treated with clofilium (Carlsson et al., 1992). In our study, EADs were also recorded by MAP catheters from rabbit ventricular epicardium and endocardium after clofilium. Our experiments were performed to mechanically stabilize the isolated heart and to ensure a stable MAP baseline, free of EADs or DADs, before collecting data. The reliability of MAP durations was corroborated by surface ECGs in these experiments. Preliminary experiments confirmed that MAPs recorded from the LV epicardium reliably reported the presence and absence of EADs in the LV endocardium. One potential disadvantage of measuring from the epicardium is that it cannot distinguish true EAD suppression by KN-93 from impaired propagation of EADs to the epicardium.
CaM kinase may be an antiarrhythmic drug target.
The results of these studies suggest the hypothesis that CaM kinase is a proarrhythmic signaling molecule for EADs. Thus, CaM kinase may be an antiarrhythmic drug target. More specific inhibitors will be needed to adequately test this hypothesis. CaM kinase is ubiquitous in mammalian cells and mediates such diverse functions as fatty acid and cholesterol metabolism, microtubule assembly, T cell activation and synaptic plasticity (Braun and Schulman, 1995a). Recent studies using transgenic mice lacking the neural-specific α isoform of CaM kinase resulted in abnormalities in synaptic plasticity (Glazewski et al., 1996), long-term potentiation (Silva et al., 1992) and spatial learning (Silva et al., 1992a,b). The diversity of CaM kinase functions presents important challenges to systemic CaM kinase inhibition. CaM kinase inhibitors could potentially serve as either primary antiarrhythmic agents in patients with long QT syndromes or supplement other antiarrhythmic regimens to reduce proarrhythmic effects inherent in drug-induced QT prolongation.
Acknowledgments
Dr. William Clusin generously provided laboratory equipment and helpful comments. Drs. Paul Bennett, Dan Roden and Dirk Snyders reviewed the manuscript and we appreciate their helpful suggestions.
Footnotes
-
Send reprint requests to: Dr. Mark E. Anderson, Division of Cardiology, Vanderbilt University School of Medicine, 315 Medical Research Building II, Nashville, TN 37232-6300.
-
This work was supported by National Institutes of Health Grant HL03727 (M.E.A.), a Cardiac Arrhythmia Research and Education Foundation, Inc. (Irvine, CA) grant (M.E.A.) the Stanford Cardiac and Cellular Electrophysiology Program Grant HL07740 from the National Institutes of Health (M.E.A.), a grant from the S.K.B. Foundation (Sunnyvale, CA) (R.J.S.) and National Institutes of Health Grant GM30179 (H.S.).
- Abbreviations:
- CaM
- calmodulin
- CaM kinase
- multifunctional Ca2+/calmodulin dependent protein kinase II
- DAD
- delayed afterdepolarization
- EAD
- early afterdepolarization
- LV
- left ventricle
- LVDP
- left ventricular developed pressure
- MAP
- monophasic action potential
- [Ca++]i
- intracellular Ca++
- ICa
- L-type Ca++ current
- Vm
- cell membrane potential KN-93, 2-[N-(2-hidroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine
- KN-9Z
- (Z-N-(4-methoxybenzenesulfonyl)-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine
- AP
- action potential
- DMSO
- dimethyl sulfoxide
- DTT
- dithiothreitol
- EGTA
- ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid
- EDTA
- ethylenediaminetetraacetic acid
- PIPES
- piperazine-N,N′-bis[z-ethanesulfonic acid]
- 1
- 4-piperazinediethanesulfonic acid
- RV
- right ventricle
- ECG
- electrocardiogram
- LVDP
- LV developed pressure
- BSA
- bovine serum albumin
- 4-AP
- 4-aminopyridine
- Received January 21, 1998.
- Accepted June 25, 1998.
- The American Society for Pharmacology and Experimental Therapeutics