Since the Cardiac Arrhythmia Suppression Trial was reported, sodium (Na⁺) channel blockers have been considered to deteriorate the prognosis of patients with cardiac arrhythmias. Sodium channel blockers decrease Na⁺ entry, which, in turn, decreases calcium (Ca²⁺) influx through L-type Ca²⁺ channels. Consequently, the decreased Ca²⁺ influx has a negative inotropic effect on cardiomyocytes. In contrast, potassium (K⁺) channel blockers (KBs) were expected to be used more frequently than before. Recent studies have demonstrated that KBs inhibit K⁺ outward current, which, in turn, causes prolongation of action potentials. Prolongation of action potentials results in a substantial increase in net Ca²⁺ entry through L-type Ca²⁺ channels, and this augmented Ca²⁺ influx enhances myocardial contractility by increasing Ca²⁺ release from the sarcoplasmic reticulum (1- 2). Potassium channel blockers, such as 4-aminopyridine (4-AP), which block K⁺ outward currents and prolong the action potential duration (APD), increase Ca²⁺ transient ([Ca²⁺]_i) in cardiomyocytes (3- 4). These data indicate that KBs could exert potentiating effects on contractility by increasing the amplitude of intracellular [Ca²⁺]_i.

Mitogen-activated protein kinase (MAPK) family proteins transduce signal from diverse receptor types, including receptor protein tyrosine kinases, G-protein-coupled receptors and cytokine receptors, and play an important role in the proliferation and differentiation of various cell types (5- 6). Among this family, extracellular signal-regulated kinase (ERK) has been reported to be activated by various cytokines and growth factors, such as phenylephrine, endothelin-1, angiotensin II and leukemia inhibitory factor (7), as well as by mechanical stretching in cardiomyocytes. The activation of ERK requires phosphorylation of both a threonine and a tyrosine residue by a dual-specificity kinase known as MEK (MAPK/ERK kinase). Activated ERK translocates into the nucleus and can phosphorylate a variety of nuclear transcription factors, suggesting that ERK is a key mediator of transduction of cytoplasmic signals to nuclear responses.

Elevation of intracellular Ca²⁺ levels can trigger various signaling events. Rosen et al. (8) reported that Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels after membrane depolarization induced activation of MEK and MAPK through activation of Ras in neurons. Moreover, Ca²⁺ influx after membrane depolarization was recently reported to mediate ligand-independent transactivation of epidermal growth factor receptor (EGFR) in PC12 cells (9- 10). Subsequently, we directed our attention to KBs and investigated whether the use of KBs can activate the ERK pathway through Ca²⁺-dependent signaling. Moreover, we also explored the upstream signals leading to ERK activation by KBs. Here, we report that KBs can induce ERK activation, which is initiated by Ca²⁺ entry through L-type Ca²⁺ channels, and that EGFR and proline-rich tyrosine 2 (Pyk2) are critically involved in KB-induced ERK activation in cardiomyocytes.

Primary cultures of cardiomyocytes were prepared from the ventricles of one-day-old neonatal Wistar rats, as described previously (11). At 48 h after seeding, the culture medium was changed to medium containing 0.5% fetal bovine serum, incubated for 6 h, and then KBs were added.

Electrophysiologic studies were performed in Tyrode’s solution with 0.5% fetal bovine serum, under various concentrations (0, 0.1 and 1 mmol/l) of 4-AP, which was dissolved in distilled water immediately before use. Cultured cells were plated on the stage of an inverted phase-contrast microscope (Diaphoto-300, Nikon, Toyko, Japan) at 37°C. Action potentials were recorded by use of a conventional microelectrode. Glass microelectrodes filled with potassium chloride (3 mol/l), with a direct current resistance of 15 to 30 MΩ, were selected. Membrane potentials were measured in the current clamp mode (MEZ-8300, Nihon Kohden, Tokyo, Japan) and stored on digital magnetic tape (frequency range 0 to 20 kHz; Sony Magnescale, Sony Co., Tokyo, Japan) for later analysis. During measurements, the cells were field-stimulated by 7-ms pulses at 1 Hz with a step-pulse generator (SET- 1201, Nihon Kohden, Tokyo, Japan).

The [Ca²⁺]_i transient was monitored by use of the fluorescent calcium indicator Fluo-4 (Molecular Probes, Eugene, Oregon), as described previously (12). During measurements, the cells were field-stimulated by 7-ms pulses at 1 Hz.

Potassium channel blockers, including 4-AP, 1-(2-[6-methyl-2-pyridil]-ethyl)-4-(4-methylsulfonylaminobenzoyl)-piperidine (E-4031, Eisai, Tokyo, Japan), tetra-ethylammonium (TEA) and quinidine, were purchased from Sigma Chemical Co., Ltd. (Tokyo, Japan). Anti-phospho-ERK antibody was purchased from New England Biolabs (Boston, Massachusetts). Anti-phosphotyrosine antibody (RC20H) was purchased from Transduction Laboratories (Lexington, Kentucky). Anti-Pyk2 and anti-EGFR antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California).

The cells were lysed with a buffer, as described previously (7). Lysates were incubated with either anti-EGFR or anti-Pyk2 antibody at 4°C for 4 h, followed by protein A- or G-sepharose (Sigma) for 2 h, and immunoprecipitated. Proteins were separated by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phosphotyrosine antibody. Western blot analysis for phospho-ERK was performed, as described previously (7). The signals were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, England).

Tranasfection of plasmids was performed using effectene transfection reagent (Qiagen, Hilden, Germany), according to the manufacturer’ s instructions. The deletion mutant (533del) of human EGFR (EGFRdel) was provided by Murasawa et al. (13). Hemaggulutinin-tagged ERK2 plasmid (HA-ERK2) with the SV40 promoter was provided by Minden et al. (14). HA-ERK2 was co-transfected with these plasmids. After 4-AP treatment, the cells were lysed, and the lysates were incubated with anti-HA antibody (Santa Cruz Biotechnology, Inc.). The immunocomplex was precipitated on protein A-sepharose, resuspended in 25 μl of kinase buffer and incubated with 25 μg of bovine myelin basic protein (Sigma) as a substrate at 30°C for 20 min. The reaction was terminated by adding Laemmli sample buffer. The supernatants were resolved by SDS-PAGE, and the gel was washed with 5% trichloroacetic acid for 30 min, dried and subjected to auto-radiography.

The data were processed using StatView J-4.5 software. All data are expressed as the mean value ± SE. Comparisons among the values of all groups (Figures 1, 2, 3, 4, 5, 6) were performed by one-way analysis of variance. For comparisons of myelin basic protein kinase activities among all groups in the transfection experiments, two-way analysis of variance was performed. The Bonferroni method was used to determine the level of significance. A p value <0.05 was considered statistically significant.

To begin to determine whether KBs can activate Ca²⁺-mediated signaling in cardiomyocytes, we needed to determine precisely how KBs can affect action potentials. 4-Aminopyridine is known to inhibit I_to and I_kur, which may culminate in the prolongation of action potentials. We first recorded the action potentials of cardiomyocytes stimulated by various concentrations of 4-AP (0, 0.1 and 1 mmol/l). (Figure 1) shows a representative tracing of the action potentials. (Figure 1) indicates the effect of 4-AP on the APD₉₀ obtained from seven separate experiments. 4-Aminopyridine prolonged APD₉₀ in a dose-dependent manner. One mmol/l of 4-AP lengthened APD₉₀ by 37%.

Prolongation of action potentials suggested that 4-AP may increase [Ca²⁺]_i in cardiomyocytes. To confirm this, we measured [Ca²⁺]_i in the cells stimulated by 4-AP (0.1 and 1 mmol/l). (Figure 2) shows a representative tracing, and (Figure 2) shows the time course of [Ca²⁺]_i of the cardiomyocytes treated with 1 mmol/l of 4-AP. 4-Aminopyridine prominently increased [Ca²⁺]_i. 4-Aminopyridine (1 mmol/l) enhanced [Ca²⁺]_i at 1 min and caused it to peak at 10 min, by 152 ± 0.5%.

The activation of ERK represents a point of convergence for growth signals originating from different type of receptors, such as G-protein-coupled receptors, tyrosine kinase receptors and cytokine receptors (6). To address the possibility that the use of KBs can affect intracellular signaling pathways through [Ca²⁺]_i in cardiomyocytes, we first investigated whether KBs can activate ERK. We incubated cardiomyocytes with either 4-AP (1 mmol/l), E4031 (2 μmol/l), TEA (10 mmol/l) or quinidine (50 μmol/l) for up to 30 min (Figure 3). Interestingly, all KBs induced phosphorylation of ERK in a time-dependent manner, which peaked at 15 min. We also stimulated the cells with leukemia inhibitory factor (LIF) as a positive control agent (Figure 3). These findings indicate that KBs could induce ERK activation to the same extent as LIF.

The KBs may affect the different K⁺ channels at different concentrations and prolong the APD at certain concentrations. To determine the dose dependency of KBs on ERK activation, we stimulated the cells with various concentrations (0.01 to 1 mmol/l) of 4-AP and detected the phosphorylation of ERK. 4-Aminopyridine phosphorylated ERK in a dose-dependent manner (Figure 3).

Next, we investigated the molecular mechanism leading to the activation of ERK by KBs. Because KBs are not physiologic ligands, and the aforementioned findings suggest that application of KBs significantly modulates intracellular Ca²⁺ in cardiomyocytes, we investigated the possible involvement of Ca²⁺ in KB-mediated ERK activation. We pre-incubated the cells with egtazic acid (1 mmol/l) and 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis (BAPTA-AM) (20 μmol/l), and we detected the phosphorylation of ERK. Interestingly, 4-AP-induced ERK activation was completely abrogated by egtazic acid and BAPTA-AM (Figure 4). Next, we investigated the involvement of the voltage-dependent L-type Ca²⁺ channel, nonselective cation channel and Na⁺/Ca²⁺ exchanger. We pre-incubated the cells with the L-type Ca²⁺ channel blocker diltiazem (2 μmol/l), the nonselective cation channel blocker flufenamic acid (100 μmol/l) and the Na⁺/Ca²⁺ exchanger blocker benzamil (100 μmol/l), and we detected ERK phosphorylation. Diltiazem almost completely inhibited the phosphorylation of ERK, whereas benzamil and flufenamic acid had no effect on this phosphorylation (Figure 4). To confirm that the augmentation of L-type Ca²⁺ current directly activates this phosphorylation, we stimulated the cells with the L-type channel opener Bayk8644 (10 μmol/l), and we detected the phosphorylation of ERK. Bayk8644 induced phosphorylation of ERK in a time-dependent manner (Figure 4). (Figure 4) shows the densitometric analysis of the mean expression of phosphorylated ERK in five separate experiments. These findings indicate that 4-AP-induced phosphorylation of ERK was mediated by voltage-dependent L-type Ca²⁺ channels.

The involvement of L-type Ca²⁺ current and Ca²⁺-induced Ca²⁺ release suggests that Ca²⁺-binding protein may take part in this ERK activation. Protein kinase C (PKC), calmodulin-dependent kinases and Pyk2 are Ca²⁺-dependent and are considered to be located upstream of ERK in various types of cells (13,15- 16). Therefore, we pre-incubated the cells with calphostin C (PKC inhibitor; 1 μmol/l), calmidazolium (calmodulin inhibitor; 50 μmol/l), KN62 (CaMKII and IV inhibitor; 10 μmol/l) and cytochalasin D (10 μmol/l), and we measured the phosphorylation of ERK. Cytochalasin D is an inhibitor of actin polymerization, and other investigators have observed that this compound can inhibit the activation of Pyk2 (17- 18). Because EGFR plays a critical role in mediating ERK activation evoked by various ligands and is considered to be parallel to or downstream of Ca²⁺-binding protein, we investigated the possible involvement of EGFR. We pre-incubated the cells with the specific EGFR inhibitor AG1478 (250 nmol/l), and we measured ERK phosphorylation. (Figure 5) shows the representative blot analysis, and (Figure 5) shows the densitometric analysis obtained from four separate experiments. Calphostin C, calmidazolium and KN62 did not inhibit the 4-AP-induced activation of ERK. However, both cytochalasin D and AG1478 strongly inhibited this phosphorylation. These findings suggest that 4-AP-induced ERK activation could be regulated through activation of EGFR and Pyk2.

To confirm whether 4-AP really activated Pyk2 or EGFR, or both, we performed immunoprecipitation-Western blot analysis to detect tyrosine phosphorylation of these two kinases. Pyk2 was phosphorylated as early as 2 min and peaked at 5 min (Figure 6). In addition, EGFR was phosphorylated as early as 5 min and peaked at 10 min after 4-AP stimulation (Figure 6). These results were reproducible in four separate experiment. These findings implicate Pyk2 and EGFR in 4-AP-induced activation of ERK in cardiomyocytes.

To confirm that EGFR is critically involved in 4-AP–induced ERK activation, we co-transfected HA-tagged ERK2 plasmid with either an EGFR deletion mutant or an empty plasmid and treated it with 4-AP. Activation of ERK was almost completely inhibited by the EGFR deletion mutant (Figure 7). These results were reproduced in four separate experiments. Taken together with the inhibitor experiment, these findings indicate that EGFR may play an important role in 4-AP-induced ERK activation.

Potassium channel blockers, which inhibit repolarization to prolong the cardiac action potentials, are commonly used for the treatment of cardiac arrhythmias. Meanwhile, K⁺ channels have been implicated in various functions in a variety of cell types (19- 21). Many agents that inhibit K⁺ channels have been shown to suppress cellular proliferation (22- 23), and KBs have been used to suppress tumor cell proliferation (19,22,24). The KBs have induced apoptosis in malignant astrocytoma cell lines, but an anti-apoptotic effect of KBs has also been reported (25). A recent report found that 2.5 mmol/l of 4-AP induced apoptosis in HepG2 cells in a Ca²⁺-dependent manner (26). This phenomenon can be explained by the fact that elevation of the intracellular Ca²⁺ concentration activates apoptosis-inducing phosphatases, Ca²⁺-dependent neutral proteinase, Ca²⁺/Mg²⁺-dependent endonuclease and Ca²⁺-dependent transglutaminase. Thus, the effect of K⁺ channel modulation on cellular function may be quite different among different cell types.

The main purpose of the present study was to investigate whether pharmacologic inhibition of K⁺ currents could influence the hypertrophic signaling cascade in the cardiomyocytes of rats. This study clearly showed that KBs evoked ERK phosphorylation, possibly through activation of Pyk2 and EGFR in cardiomyocytes, initiated by the entry of Ca²⁺ through L-type Ca²⁺ channels. There are several lines of evidence to suggest that the ERK pathway plays an important role in mediating cardiac hypertrophy (5- 7). Various hypertrophic stimuli can induce activation of the ERK pathways. However, Thorborn et al. (27) reported that overexpression of the active forms of Raf-1 and ERK does not cause the sarcomeric organization typical of hypertrophic growth, and Post et al. (28) reported that inhibition of MEK by PD98059 did not suppress phenylephrine-induced sarcomeric organization or atrial natriuretic peptide gene expression. Moreover, they showed that adenosine triphosphate and carbachol activated the Raf-1/MEK/ERK cascade, although neither of these reagents could cause cardiac hypertrophy. These findings indicate that activation of ERK alone was not sufficient for the induction of cardiac hypertrophy and hypertrophic gene expression, as well as suggest that the role of this pathway was distinct from ligand to ligand. The KBs are frequently used for the treatment of cardiac arrhythmias and have never been reported to induce cardiac hypertrophy in vivo. The effect of KBs on the hypertrophy-mediating pathway in cardiomyocytes remains unknown; however, it is possible that activation of ERK by KBs does not directly cause cardiac hypertrophy.

In this study, we used four different kinds of KBs. 4-Aminopyridine inhibits I_to and I_kur, but not I_kr and I_ks. Tetra-ethylammonium inhibits I_kr, I_kur and, partially, I_ks, but not I_to. E4031 only inhibits I_kr in cultured cardiomyocytes (29). Quinidine blocks I_kur, I_kr, I_ks (>300 μmol/l) and I_to(30). Although they have different target channels, all of these agents can prolong the APD. Because these agents have different chemical structures, are not physiologic ligands and have different target channels, the mechanism of ERK activation may be caused by their common effect of prolongation of action potentials. We suspected that inhibition of specific K⁺ currents might not correlate with KB-induced ERK activation.

The present study demonstrated that Ca²⁺-mediated signaling was critically involved in 4-AP-induced ERK activation. Elevated intracellular levels of Ca²⁺ can trigger various signaling events, resulting in stimulation of the MAPK pathway. In PC12 cells, Ca²⁺ influx after membrane depolarization by potassium chloride mediates ligand-independent phosphorylation of EGFR in both a CaMKII-dependent and -independent manner (10,31). Pyk2 is activated in PC12 cells by extracellular stimuli that increase intracellular Ca²⁺ in either a PKC-dependent or -independent manner (16). Calmodulin-dependent kinases are activated by an increase in intracellular Ca²⁺ levels mediated by Ca²⁺ influx through L-type Ca²⁺ channels (32- 33) or by Ca²⁺ release from the intracellular store (34). Therefore, we examined the downstream pathway of 4-AP-induced augmentation of intracellular Ca²⁺ leading to ERK activation. The present study demonstrated that Pyk2 and EGFR were involved in this activation of ERK (Figure 8). Because 4-AP only increases intracellular Ca²⁺ levels, we suspected that 4-AP-induced activation of Pyk2 may be mediated, not by PKC, but only by Ca²⁺. The molecular mechanism of the activation of EGFR by 4-AP remains unknown, but we supposed that Pyk2 might be involved in EGFR activation, based on the finding that cytochalasin D, but not calmidazolium, calphostin C nor KN62, inhibited the activation of ERK.

The KBs are known to prolong QT intervals in the clinical setting, and many reports have focused on the electrophysiologic side effects of high concentrations of KBs, such as torsade de pointes and even fatal arrhythmias. To date, however, no clinical or experimental studies have examined the biologic effect of these compounds in cardiomyocytes. The present study has demonstrated that, when we consider the side effects of KBs, we should bear in mind not only the electrophysiologic effects, but also the molecular effects, through various signaling pathways. Further studies are required to determine the effect of KBs on the cellular function in cardiomyocytes.