Potassium channel blocker activates extracellular signal-regulated kinases through Pyk2 and epidermal growth factor receptor in rat cardiomyocytes
Satoko Tahara, MD*,
Keiichi Fukuda, MD, PhD*, ,
Hiroaki Kodama, MD*,
Takahiro Kato, MD*,
Shunichiro Miyoshi, MD, PhD and
Satoshi Ogawa, MD, PhD*
* Cardiopulmonary Division, Department of Internal Medicine, Tokyo, Japan
Institute for Advanced Cardiac Therapeutics, Tokyo, Japan
Department of Physiology, Keio University School of Medicine, Tokyo, Japan
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Figure 1 4-Aminopyridine (4-AP) prolonged the action potential duration (APD) in cardiomyocytes. Action potentials of the cardiomyocytes were recorded as described in the Methods section, using a conventional glass micropippete, and the cells were treated with 4-AP (0.1 and 1 mmol/l) for 5 min. (A) Representative tracings of the action potentials. These tracings were obtained from the same cells. (B) Effect of 4-AP on the APD90. The experiments were repeated three times, and the data are presented as the mean value ± SE obtained from 15 different cells. 4-Aminopyridine increased the APD by 37% at 1 mmol/l. *p < 0.01 vs. control cells. NS = not significant.
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Figure 2 4-Aminopyridine (4-AP) enhances calcium transient [Ca2+]i in cardiomyocytes. Cardiomyocytes were pretreated with Fluo-4 and treated with 4-AP for the indicated times. [Ca2+]i was recorded as described in Methods. (A) Representative tracings of [Ca2+]i. The tracings were recorded from the same cells. The cells were treated with 1 mmol/l of 4-AP for 10 min. (B) The time course of [Ca2+]i. One mmol/l of 4-AP increased [Ca2+]i by 52% at 10 min. The experiments were repeated three times, and the data are presented as the mean value ± SE obtained from 15 different cells. *p < 0.01 and **p < 0.05 vs. control cells.
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Figure 3 Potassium channel blockers activate extracellular signal-regulated kinase (ERK) in cardiomyocytes. Cardiomyocytes were treated with (A) 4-aminopyridine (4-AP) (1 mmol/l), (B) E4031 (2 µmol/l), (C) tetraethylammonium (TEA) (10 mmol/l) and (D) quinidine (50 µmol/l) for the indicated times, and phosphorylation of ERK was detected by Western blot analysis. All potassium channel blockers induced phosphorylation of ERK, which peaked at 15 min. (E) Leukemia inhibitory factor (LIF) was used as a positive control agent. (F) 4-Aminopyridine caused dose-dependent augmentation of the phosphorylation of ERK in cardiomyocytes. The cells were treated with 0.01, 0.1 and 1 mmol/l of 4-AP for 15 min. All experiments were repeated at least four times and gave similar results.
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Figure 4 Involvement of calcium (Ca2+) and L-type Ca2+ channels in 4-aminopyridine (4-AP)-induced ERK activation. (A) Cardiomyocytes were pretreated with ethyleneglycoltetraacetic acid (EG) (1 mmol/l) or BAPTA-AM (BAP) (20 µmol/l) for 30 min and treated with 4-AP (1 mmol/l) for 15 min. Both EG and BAPTA-AM completely abolished 4-AP-induced extracellular signal-regulated kinase (ERK) phosphorylation. (B) The cells were pretreated with the L-type Ca2+ channel blocker diltiazem (Dil) (2 µmol/l) and the Na+/Ca2+ exchanger inhibitor benzamil (BZ) (100 µmol/l) for 5 min, and then treated with 4-AP for 15 min. Diltiazem fully inhibited ERK phosphorylation, whereas BZ did not. (C) The cells were pre-incubated with the nonselective cation channel blocker fulfenamic acid (FA) (100 µmol/l) and then treated with 4-AP for 15 min. Fulfenamic acid was dissolved in ethanol (vehicle). Fulfenamic acid did not attenuate 4-AP-induced ERK activation. (D) The cells were treated with the L-type Ca2+ channel opener Bayk8644 (10 µmol/l) for the indicated times. BayK8644 induced phosphorylation of ERK. All experiments were repeated at least five times, and we obtained similar results. (E) Densitometric analysis of the phosphorylation of ERK was shown. Data are presented as the mean value ± SE. *p < 0.01 vs. 4-AP alone. LIF = leukemia inhibitory factor; NS = not significant.
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Figure 5 Involvement of epidermal growth factor receptor (EGFR) and Pyk2 in 4-aminopyridine (4-AP)-induced extracellular signal-regulated kinase (ERK) activation. (A, B) Cardiomyocytes were pre-incubated with either KN62 (KN [calmodulin kinase II and IV inhibitor]) (10 µmol/l), AG1478 (AG [epidermal growth factor inhibitor]) (250 nmol/l), cytochalasin D (CyD [actin dimerization inhibitor]) (10 µmol/l), calphostin C (Cal [PKC inhibitor]) (1 µmol/l) or calmidazolium (CMZ [calmodulin inhibitor]) (50 µmol/l) for 30 min and then treated with 4-AP for 15 min. Leukemia inhibitory factor (LIF) was used as a positive control. AG1478 and cytochalasin D strongly blocked 4-AP-induced ERK activation, whereas the other agents did not. (C) Densitometric analysis of the inhibitor experiment. Representative blot analyses are shown. All experiments were repeated at least five times and yielded similar results. Data are presented as the mean value ± SE. *p < 0.01 vs. 4-AP alone.
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Figure 6 4-Aminopyridine (4-AP) phosphorylates proline-rich tyrosine kinase 2 (Pyk2) (A) and epidermal growth factor receptor (EGFR) (B) in cardiomyocytes. Cardiomyocytes were stimulated with 4-AP for the indicated times, and the tyrosine phosphorylation of Pyk2 and EGFR was detected by immunoprecipitation-Western blot analysis. Endothelin (ET-1) was used as a positive control agent. Anti-p-Tyr represents anti-phosphotyrosine antibody. 4-Aminopyridine induced phosphorylation of Pyk2 and EGFR at 5 and 10 min, respectively, in cardiomyocytes.
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Figure 7 Effect of epidermal growth factor receptor (EGFR) deletion mutant on 4-aminopyridine (4-AP)-induced extracellular signal-regulated kinase (ERK) activation in cardiomyocytes. (Top) Cardiomyocytes were co-transfected with HA-tagged ERK2 and either deletion mutant of EGFR (pDN-EGFR), or empty plasmid (pcDNA), and treated with 4-AP for 15 min. The immunocomplex with anti-HA antibody was precipitated on protein A-sepharose, and in vitro myelin basic protein (MBP) kinase assay by ERK2 was performed, as described in Methods. Data are presented as the mean value ± SE of the results of five separate experiments determined by densitometry. (Bottom) Densitometric analysis of the inhibitory effect of dominant-negative EGFR on 4-AP-induced myelin basic protein kinase activity of ERK. Data are presented as the mean value ± SE. *p < 0.01 vs. 4-AP–treated cells transfected with the empty vector. pDN-EGFR = dominant negative EGFR plasmid.
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Figure 8 Model of 4-aminopyridine (4-AP)-induced extracelluarl signal-regulated kinase (ERK) activation in cardiomyocytes. Treatment with 4-AP results in augmentation of L-type calcium (Ca2+) current and calcium transient ([Ca2+]i), leading to the activation of proline-rich tyrosine kinase 2 (Pyk2) and EGFR. Transactivation of EGFR predominantly contributes to ERK activation. EGTA = ethyleneglycoltetraaceticacid.
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