Author + information
- Received November 29, 2000
- Revision received June 27, 2001
- Accepted July 19, 2001
- Published online November 1, 2001.
- Satoko Tahara, MD*,
- Keiichi Fukuda, MD, PhD*,† (, )
- Hiroaki Kodama, MD*,
- Takahiro Kato, MD*,
- Shunichiro Miyoshi, MD, PhD‡ and
- Satoshi Ogawa, MD, PhD*
- ↵*Reprint requests and correspondence: Dr. Keiichi Fukuda, Institute for Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
We sought to determine whether potassium (K+) channel blockers (KBs) can activate extracellular signal-regulated kinase (ERK) and to characterize the upstream signals leading to ERK activation in cardiomyocytes.
Because KBs attenuate K+outward current, they may possibly prolong the duration of action potentials, leading to an increase in calcium (Ca2+) transient ([Ca2+]i) in cardiomyocytes. Elevation of intracellular Ca2+levels can trigger various signaling events. Influx of Ca2+through L-type Ca2+channels after membrane depolarization induced activation of MEK and ERK through activation of Ras in neurons. Although KBs are frequently used to treat cardiac arrhythmias, their effect on signaling pathways remains unknown.
Primary cultured rat cardiomyocytes were stimulated with four different KBs—4-aminopyridine (4-AP), E-4031, tetra-ethylammonium and quinidine—and phosphorylation of ERK, proline-rich tyrosine kinase 2 (Pyk2) and epidermal growth factor receptor (EGFR) was detected. Action potentials were recorded by use of a conventional microelectrode. (Ca2+)iwas monitored by the fluorescent calcium indicator Fluo-4.
E-4031, 4-AP, tetra-ethylammonium and quinidine induced phosphorylation of ERK. 4-Aminopyridine prolonged the duration of action potentials by 37% and increased (Ca2+)iby 52% at 1 mmol/l. Pre-incubation of ethyleneglycoltetraacetic acid, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis and diltiazem completely blocked this phosphorylation, whereas flufenamic acid and benzamil did not. 4-Aminopyridine induced tyrosine phosphorylation of Pyk2 and EGFR, which peaked at 5 and 10 min, respectively. Cytochalasin D, AG1478 and dominant-negative EGFR strongly inhibited the phosphorylation of ERK, whereas calphostin C, calmidazolium and KN62 did not.
These findings indicate that KBs induce ERK activation, which starts with Ca2+entry through the L-type Ca2+channel in cardiomyocytes, and that EGFR and Pyk2 are involved in this activation.
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.
Recording of action potentials
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).
Measurement of [Ca2+]i
The [Ca2+]itransient 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.
Reagents and antibodies
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).
Immunoprecipitation and Western blot analysis
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).
Transfection of plasmids and in vitro kinase assay
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 (Figs. 1 to 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.
4-Aminopyridine prolongs the APD in cardiomyocytes
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 Itoand Ikur, 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 1Ashows a representative tracing of the action potentials. Figure 1Bindicates the effect of 4-AP on the APD90obtained from seven separate experiments. 4-Aminopyridine prolonged APD90in a dose-dependent manner. One mmol/l of 4-AP lengthened APD90by 37%.
4-Aminopyridine increases intracellular [Ca2+]i
Prolongation of action potentials suggested that 4-AP may increase [Ca2+]iin cardiomyocytes. To confirm this, we measured [Ca2+]iin the cells stimulated by 4-AP (0.1 and 1 mmol/l). Figure 2Ashows a representative tracing, and Figure 2Bshows the time course of [Ca2+]iof the cardiomyocytes treated with 1 mmol/l of 4-AP. 4-Aminopyridine prominently increased [Ca2+]i. 4-Aminopyridine (1 mmol/l) enhanced [Ca2+]iat 1 min and caused it to peak at 10 min, by 152 ± 0.5%.
KBs activate ERK in cardiomyocytes
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+]iin 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 (Fig. 3A to 3D). 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 (Fig. 3E). 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 (Fig. 3F).
Involvement of an L-type Ca2+channel-mediated increase in [Ca2+]ion KB-mediated ERK activation
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 (Fig. 4A). 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 (Fig. 4B and 4C). 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 (Fig. 4D). Figure 4Eshows 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.
Involvement of Pyk2 and EGFR in K+channel blocker-induced ERK activation
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 5A and 5Bshows the representative blot analysis, and Figure 5Cshows 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.
4-Aminopyridine induces phosphorylation of Pyk2 and EGFR
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 (Fig. 6A). In addition, EGFR was phosphorylated as early as 5 min and peaked at 10 min after 4-AP stimulation (Fig. 6B). These results were reproducible in four separate experiment. These findings implicate Pyk2 and EGFR in 4-AP-induced activation of ERK in cardiomyocytes.
Deletion mutant of EGFR inhibits 4-AP-induced ERK activation
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 (Fig. 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.
K+channel modulation and cellular function
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.
KB induces ERK activation
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.
Relationship between ERK activation and specific potassium currents
In this study, we used four different kinds of KBs. 4-Aminopyridine inhibits Itoand Ikur, but not Ikrand Iks. Tetra-ethylammonium inhibits Ikr, Ikurand, partially, Iks, but not Ito. E4031 only inhibits Ikrin cultured cardiomyocytes (29). Quinidine blocks Ikur, Ikr, Iks(>300 μmol/l) and Ito(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.
Involvement of EGFR and Pyk2 in 4-AP-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 (Fig. 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.
☆ This study was supported by research grants from the Ministry of Education, Science and Culture, Japan, and Health Science Research Grants for Advanced Medical Technology from the Ministry of Welfare, Japan.
- action potential duration
- 1,2-bis(2-aminophenoxy)-ethane- N,N,N′,N′-tetraacetic acid tetrakis
- epidermal growth factor receptor
- extracellular signal-regulated kinase
- potassium channel blocker
- leukemia inhibitory factor
- mitogen-activated protein kinase
- MAPK/ERK kinase
- protein kinase C
- proline-rich tyrosine 2
- Received November 29, 2000.
- Revision received June 27, 2001.
- Accepted July 19, 2001.
- American College of Cardiology
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