Potassium channel blocker activates extracellular signal-regulated kinases through Pyk2 and epidermal growth factor receptor in rat cardiomyocytes

OBJECTIVES 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. BACKGROUND 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. METHODS 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+)i was monitored by the fluorescent calcium indicator Fluo-4. RESULTS 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+)i by 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. CONCLUSIONS 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.