• 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • br Conflict of Interest br Acknowledgments br


    Conflict of Interest
    Introduction Human ether-a-go-go-related gene (hERG) encodes the alpha subunit of the rapidly-activating delayed-rectifier K+ channel, which plays a pivotal role in repolarization of cardiac action potentials (APs) [1]. Most of mutant hERG proteins causing the type 2 long QT syndrome (LQT2) fail to mature in the endoplasmic reticulum (ER) and Golgi apparatus [2]. Because of their instability, they are degraded through the ubiquitin proteasome system (UPS), resulting in reductions of hERG protein expression on the cell membrane and channel currents [3]. A possible way to rescue mutant hERG proteins is the use of a chemical chaperone, which is defined as a small molecule that can bind to a protein and stabilize it [4]. Several agents have been reported to bind to hERG proteins inside the cell and restore their stability, including hERG activators [5] and hERG blockers [6,7]. HERG channel blockers, such as E4031, fexofenadine and astemizole, facilitated maturation of hERG proteins by acting as chemical chaperones [8]; however, these agents cannot be used clinically for enhancing hERG channel currents, because they acutely block the rapidly-activating delayed-rectifier K+ channel current (I). Pilsicainide (C17H24N2O) is a widely used antiarrhythmic agent, especially for treating atrial fibrillation [9]. It belongs to the class Ic antiarrhythmic agent (Na+ channel blocker) as classified by Vaughan Williams [10], reducing the maximum rate of AP upstroke in atria, ventricles and Purkinje fibers [11]. The effects of pilsicainide on AP repolarization are controversial: Pilsicainide did not influence AP EVP-6124 Supplier repolarization in rat ventricular myocytes [12]. However, pilsicainide has been reported to block hERG channel currents expressed in HEK293 EVP-6124 Supplier [13], implying the reduction of I and prolongation of AP duration (APD) by the agent. In contrast, pilsicainide shortened APDs in canine Purkinje fibers [11] and in guinea pig ventricular myocardia [14], which might reflect the enhancement of I via stabilization of hERG proteins. However, it remains unknown whether pilsicainide actually stabilizes hERG proteins. In the present study, we studied the effects of acute and chronic exposures to pilsicainide on the expression of hERG proteins and found that chronic treatment with pilsicainide as a chemical chaperon could stabilize hERG proteins in transfected mammalian cells.
    Materials and methods
    Discussion In the present study, chronic treatment with pilsicainide stabilized WT-hERG proteins, increased WT-hERG channels in the plasma membrane, and thereby enhanced the hERG channel current I. Pilsicainide exerted these effects inside the cell via an action as a chemical chaperone, independently of phosphorylation of Akt and enhanced expression of HSF-1, hsp70 or hsp90. Pilsicainide is a class Ic antiarrhythmic agent that blocks Na+ channels and reduces the maximum rate of AP upstroke. Pilsicainide occasionally caused QT prolongation, suggesting its inhibitory action on outward K+ currents. Wu et al. have reported that, although pilsicainide barely affected K+ and Ca2+ currents at therapeutic concentrations of 0.20–0.90μg/mL (0.73–3.31μM), it blocked hERG channel currents at concentrations higher than the therapeutic range [13]. In the present study, chronic treatment with pilsicainide at therapeutic concentrations significantly increased WT-hERG protein expressions via the prolongation of the half-life of WT-hERG proteins and thereby enhanced hERG channel currents, whereas acute treatment with pilsicainide at 10μM or less influenced neither hERG protein expressions nor channel currents. Thus, while pilsicainide has been observed to cause QT prolongation occasionally, it can also shorten QT intervals by enhancing I. It is well known that Na+ channel blockers bind to the Na+ channel from inside of the cell but the charged form blocker cannot penetrate the plasma membrane [16]. In the present study, the membrane-impermeant form of pilsicainide (N-Me-PIL-MeSO4) did not increase the protein level of WT-hERG, suggesting that pilsicainide normally penetrates the plasma membrane and induces post-translational modifications of the WT-hERG protein.